Porin B (PorB) as a therapeutic target for prevention and treatment of infection by Chlamydia

ABSTRACT

The present invention features peptides of a PorB polypeptide, which PorB peptides are useful in production of antibodies that bind the full-length PorB polypeptide and as a therapeutic agent. In specific embodiments the invention features a composition comprising one or more PorB peptides (other than a full-length PorB polypeptide), which peptides contain at least one epitope that can elicit  Chlamydia -neutralizing antibodies. The invention also features methods for induction of a protective immune response against infection by  Chlamydia  and  Chlamydiophila.

This application is a Divisional application of application Ser. No.10/094,407, filed Mar. 7, 2002, now U.S. Pat. No. 7,105,171.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made, at least in part, with a government grant fromthe National Institutes of Health (Grant Nos. NIH grants AI40250,AI39258, and AI42156). Thus, the U.S. government may have certain rightsin this invention.

FIELD OF THE INVENTION

The invention relates generally to the field of treatment, prevention,and diagnosis of infectious disease, particularly to prevention ofinfectious disease caused by the bacterial pathogen Chlamydia andChlamydophila (formerly classified as, for example, C. psittacci and C.pneumoniae).

BACKGROUND OF THE INVENTION

Chlamydiae are obligate intracellular pathogens that cause a spectrum ofdiseases including trachoma, the leading cause of preventable blindnessworldwide, as well as a variety of sexually transmitted diseases such aslymphogranuloma venereum, urethritis, cervicitis, endometritis, andsalpingitis (Thylefors et al. (1995) Bull W H O 73:115-121). Forexample, Chlamydia trachomatis is considered the world's most commonsexually transmitted bacterial pathogen (Schachter and Grayston (1998)Presented at the Ninth international symposium on human chlamydialinfection, Napa, Calif.; World Health Organization, 1996, Globalprevalence and incidence of selected curable sexually transmitteddiseases: overview and estimates). Currently an estimated 400 millionpeople have active infectious trachoma, while 90 million have a sexuallytransmitted disease caused by C. trachomatis (World Health Organization,1996). Chlamydia pneumoniae usually infects the lungs and causes no morethan a mild cold; however, it can travel to the blood vessels and thrivein clots, causing heart disease. Diseases caused by Chlamydia representsignificant health problems worldwide.

Growth of Chlamydia generally depends on the acquisition of host ATP andother high-energy metabolites from the host (Moulder et al. (1991)Microbiol. Rev. 55:143-90). Chlamydiae have the enzymatic machinery forthe Embden-Meyerhoff pathway (EMP), the pentose phosphate pathway.(PPP), and the tricarboxylic acid (TCA) cycle (Kalman et al. (1999) Nat.Genet. 21:385-9; Stephens et al. (1998) Science 282:754-9). The TCA inchlamydia is incomplete in that the host lacks three enzymes: citratesynthase, aconitase, and isocitrate dehydrogenase (Kalman et al., ibid,;Stephens et al., ibid.). This observation suggests that the glutamateand α-ketoglutarate are obtained from the host cell since these cannotbe synthesized by the bacterium. It has been shown that chlamydiaeutilize glucose as the major source of carbon, but that dicarboxylatesalso serve to support chlamydial viability and growth (Iliffe-Lee et al.(2000) Mol. Microbiol. 38:20-30).

Treatment for Chlamydia infection typically involves administration ofan antimicrobial drug such as azithromycin, doxycycline, ofloxacin,erythromycin, or amoxicillin (Centers for Disease Control andPrevention. Recommendations for the prevention and management ofChlamydia trachomatis infections. Morb Mortal Wkly Rep 1993; 42 (RR-12):1-102). These conventional treatments are problematic for severalreasons, including patient non-compliance with multi-day, multi-doseregimens and side effects such as gastrointestinal problems.Furthermore, treatment of Chlamydia with existing antimicrobial drugsmay lead to development of drug resistant bacterial strains,particularly where the patient is concurrently infected with othercommon bacterial infections.

In addition, chlamydial infections often have no overt symptoms, soirreversible damage can be done before the patient is aware of theinfection. Therefore, prevention of the infection is considered the bestway to protect from the damage caused by Chlamydia. Therefore, thedevelopment and production of effective chlamydial vaccines, moreeffective treatments once infection is established, and sensitive andspecific diagnostic assays are important public health priorities.

Chlamydia have a unique developmental growth cycle with morphologicallydistinct infectious and reproductive forms, elementary bodies (EB) andreticulate bodies (RB), respectively. The outer membrane proteins of EBare highly cross-linked with disulfide bonds. The chlamydial outermembrane complex (COMC), which includes the major outer membrane protein(MOMP), is a major component of the chlamydial outer membrane. The COMCis made up of a number of cysteine-rich proteins (Everett et al. (1995)J. Bacteriol. 177:877-882; Newhall et al. (1986) Infect. Immun.55:162-168; Sardinia et al. (1988) J. Gen. Microbiol. 134:997-1004), asdetermined by the insolubility of proteins in the weak anionic detergentN-lauryl sarcosinate (Sarkosyl). Insolublity of proteins in Sarkosyl isa characteristic of integral outer membrane proteins of gram-negativebacteria (Filip et al. (1973) J. Bacteriol. 115:717-722). The COMC ispresent on the outer membrane proteins of EB, but not of RB. Incontrast, MOMP is present throughout the developmental cycle in both EBand RB and is thought to have a structural role due to its predominanceand extensive disulfide crosslinking in the EB membrane. Anotherfunction of MOMP is as a porin which allows for non-specific diffusionof small molecules into Chlamydia (Bavoil et al. (1984) Infect. Immun.44:479-485, Wyllie et al. (1998) Infect. Immun. 66:5202-5207).

As with many pathogens, the development of a vaccine to Chlamydia hasproven difficult. Much of the focus for a vaccine candidate has been onthe chlamydial major outer membrane protein (MOMP) (see, e.g., U.S. Pat.Nos. 5,770,714 and 5,821,055; and PCT publication nos. WO 98/10789; WO99/10005); WO97/41889 (describing fusion proteins with MOMPpolypeptides); WO98/02546 (describing DNA immunization based onMOMP-encoding sequences); WO 94/06827 (describing synthetic peptidevaccines based on MOMP sequences); WO 96/31236). MOMP has been estimatedto make up over 60% of the total outer membrane of Chlamydia and is anexposed surface antigen (Caldwell et al. (1981) Infect. Immun.31:1161-1176) with different sequence regions conferring serotype,serogroup and species reactivities (Stephens et al. (1988) J. Exp. Med.167:817-831). The protein consists of five conserved segments and fourvariable segments with the variable segments corresponding to surfaceexposed regions and conferring serologic specificity (Stephens et al.(1988) J. Exp. Med. 167:817-831). It has been suggested that thesevariable segments provide Chlamydia with antigenic variation, which inturn is important in evading the host immune response (Stephens, 1989“Antigenic variation of Chlamydia trachomatis,” p. 51-62. In J. W.Moulder (ed.), Intracellular Parasitism. CRC Press, Boca Raton.). Apotential problem in making a vaccine to an antigenically variant regionis that a vaccine to one region of MOMP may only confer protection tothat serovar. Also, making a subunit vaccine to an antigenic variableregion may prove difficult since conformational antigenic determinantsmay be essential to elicit effective immunization (Fan et al. (1997) J.Infect. Dis. 176:713-721). Although the use of MOMP as a vaccine stillseems promising, these potential problems strongly suggest that othervaccine targets should be explored.

Other proposed Chlamydia vaccine targets have been described andinclude, for example, glycolipid exoantigen (see, e.g., U.S. Pat. Nos.5,840,297; 5,716,793 and 5,656,271). Other Chlamydia vaccines have usedother proteins (see, e.g, PCT publication no. WO 98/58953, describing asurface protein of C. pneumoniae) or a cocktail of proteins (see, e.g.,U.S. Pat. Nos. 5,725,863; and 5,242,686) or have used live or attenuatedwhole bacteria (see, e.g., U.S. Pat. Nos. 5,972,350; 4,267,170; and4,271,146). The sequencing of the genome of C. trachomatis has provideda tool to identify candidate vaccine targets (Stephens et al. (1998)Science 282:754-759) and examination of antibodies present in serum ofinfected individuals (Sanchez-Campillo et al. (1999) Electrophoresis20:2269-79) have provided tools for the identification of additionalvaccine targets.

There is a need in the field for the development of chemotherapeuticsand vaccines that provide protection against Chlamydia andChlamydiophila infection. The present invention addresses these needs.

SUMMARY OF THE INVENTION

The present invention features peptides of a PorB polypeptide, whichPorB peptides are useful in production of antibodies that bind thefull-length PorB polypeptide and as a therapeutic agent. In specificembodiments the invention features a composition comprising one or morePorB peptides (other than a full-length PorB polypeptide), whichpeptides contain at least one epitope that can elicitChlamydia-neutralizing antibodies. The invention also features methodsfor induction of a protective immune response against infection byChlamydia and Chlamydiophila.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an alignment of the amino acid sequences of PorB (SEQ ID NO:2)and MOMP (SEQ ID NO:3).

FIG. 2 is a schematic showing the alignment of the amino acid sequencesof PorB from C. trachomatis serovars D (CT-D) (SEQ ID NO:2), L2 (CT-L2)(SEQ ID NO:5), and C (CT-C) (SEQ ID NO:6), as well as the amino acidsequence of PorB from C. pneumoniae (CPn) (SEQ ID NO:4). C. trachomatisserovar L2 and C differences are indicated below the amino acidsequence. The cysteines are indicated with an asterisk above the aminoacid sequence.

FIG. 3 is a graph showing antibody neutralization of C. trachomatisserovar L2 in HaK cells. Results are expressed as percentage reductionin inclusion-containing cells with respect to number ofinclusion-containing cells observed after incubation with SPG only. Theantibodies used were anti-PorB (open circles), anti-PorB²⁴⁻⁷¹ (closedtriangles), IH5 (closed circles), anti-pgp3 (open squares) andpre-immune serum (closed squares).

FIGS. 4A and 4B are graphs showing the results of a liposome swellinganalysis of PorB (FIG. 4A) and the outer membrane of E. coli expressingMOMP (panel B). Liposomes were made as described and 0.017 ml out of atotal of 0.3 ml was diluted in 0.6 ml of isotonic sugar solutions ofstachyose (closed circles), sucrose (open squares), glucose (closedtriangles) and arabinose (open circles). The y-axis represents a rangeof A₄₀₀ 0.15.

FIGS. 5A-5B are graphs showing lack of amino acid transport throughPorB. Liposome swelling analysis of PorB (panel A) and the outermembrane of E. coli expressing MOMP (panel B). Liposomes were made asdescribed and 0.017 ml out of a total of 0.3 ml was diluted in 0.6 ml ofisotonic sugar solutions of stachyose, arabinose (open circles) andalanine (closed triangles). The y-axis represents a range of A₄₀₀ 0.15.

FIGS. 6A and 6B are graphs showing the results of a liposome swellinganalysis of PorB (panel A) and the outer membrane of E. coli expressingMOMP (OmpA) (panel B). Isotonic solutions of stachyose (closed circles),arabinose (closed diamonds) and α-ketoglutarate (open triangles). They-axis represents a range of A₄₀₀ 0.15.

FIG. 7 is a graph showing the results of a liposome swelling assay tocontrol for effects of ions that may be present in the test solute.Liposomes containing NAD⁺, stachyose, and imidazone-NAD were diluted inisotonic test solutions of citrate (closed circles), oxaloacetate(closed diamonds), and α-ketoglutarate (open triangles). The y-axisrepresents a range of A₄₀₀ 0.15.

FIG. 8 is a graph showing enzyme-linked liposome assay testing of theentry and oxidation of α-ketoglutarate. The formation of NADH usingliposomes containing PorB (closed circles) or lacking PorB (closedsquares) was measured by an increase in O.D.₃₄₀.

FIG. 9 is a graph showing liposome swelling analysis of PorB usingTCA-cycle intermediates. Isotonic sugar solutions of stachyose (closedcircles), arabinose (closed diamonds), α-ketoglutarate (open triangles),malate (closed squares) and succinate (open circles). The y-axisrepresents a range of A₄₀₀ 0.15.

FIG. 10 is a schematic showing the structures of compounds tested fordiffusion into liposomes containing PorB. Compounds in shaded boxes werenot efficiently transported by PorB.

FIG. 11. is a graph showing the results of epitope mapping of PorBprotein. Polyclonal immune sera were used to probe overlapping syntheticpeptides representing the entire PorB sequence. Each vertical barrepresents the mean absorbance (±SEM) of a peptide in thepeptide-specific ELISA. Reactivities for pooled rabbit antisera raisedagainst purified chlamydial EB (Panel A), pooled sera from humansinfected with C. trachomatis (Panel B) and sera from mice immunized withpurified PorB (Panel C) are shown. Sera were tested at a 1:1000dilution.

FIG. 12 is a graph showing the accessibility of PorB epitopes on thesurface of chlamydial EB, as determined by pre-incubation of purifiedchlamydial EB with mouse antisera specific to the peptides and detectionof residual reactivity to homologous peptide. The results are presentedas % reduction in serum reactivity (A₄₉₂) to PorB peptides of theinvention due to absorption with viable EB.

FIG. 13 is a graph showing the change in reactivities of EB-absorbedpolyclonal immune sera to PorB peptides. Rabbit, human and mouse immunesera were pre-incubated with chlamydial EB and assayed for residualreactivity to PorB polypeptides of the of the present invention byELISA. The results are presented as % reduction in serum reactivity(A₄₉₂) due to absorption with viable EB. Sera were tested at a 1:1000dilution.

FIG. 14 is an image of a dot-blot analysis to determine the specificityand surface reactivity of PorB peptide antisera. Peptide-specificantisera were used to probe viable (Row A) or SDS-treated (Row B)chlamydial EB immobilized on a nitrocellulose membrane. Peptide antiseraand pre-immune negative control sera were tested at a 1:100 dilution.The Pgp3-specific control antisera was used at a 1:500 dilution and thepositive control monoclonal to MOMP, 2C5 was tested at a 1:2000dilution.

FIG. 15 is a graph showing the results of assays to detectneutralization of infectivity for C. trachomatis EB with serialdilutions of PorB peptide antisera. Sera titer representing 50%neutralization of infectivity (±SEM) for C. trachomatis EB are shown forthe various PorB peptide antisera. EB were incubated with serialdilutions of peptide antisera and added to HaK cells. Inclusion formingunits (IFU) were dilutions counted and the % neutralization for eachdilution relative to the diluent SPG control was determined.

FIG. 16 is a schematic representing a model of secondary structure forC. trachomatis PorB protein (SEQ ID NO: 47). Peptides exposed on thesurface of chiamydial EB as determined by absorption ELISA and dot-blotanalysis and peptides that elicit neutralizing antibody responses areindicated by bars below.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “thepolynucleotide” includes reference to one or more polynucleotides andequivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

As a point of clarification, it is noted that recently two species ofbacterium, Chlamydia psittacci and Chlamydia pneumoniae, have beenre-classified into the genus Chlamydophila. Unless specifically notedotherwise, reference to the genus Chlamydia is meant to encompass allbacteria belonging to this genus, as well as the psittacci andpneumoniae species that have or soon may be reclassified asChlamydophila. Use of the terms “Chlamydia” and “chlamydial” are notmeant to be limiting to those bacterial species originally classifiedChlamydia, but are also meant to encompass the newly classified speciesof Chlamydophila as well unless specifically noted otherwise.

As used herein, “immunogenic”, as in the context of “immunogenicpeptide”, means that the peptide induces an immune response in the host(such as an antibody response) in the absence of a carrier protein. Asused herein “antigenic”, as in the context of an “antigenic peptide”,means that the peptide can be bound by antigen-specific antibody.Antigenic peptides may be made to be immunogenic by coupling the peptideto a carrier and/or by administration with an adjuvant. An “immunogeniccomposition” thus can comprise, for example, an immunogenic peptide, oran antigenic peptide and an adjuvant or carrier. Immunogeniccompositions of the invention are useful for the production of anti-PorBpolypeptide antibodies and as the basis for vaccines.

As used herein, “immunoprotective response” is meant to encompasshumoral and/or cellular immune responses that are sufficient to: 1)inhibit or prevent infection by a microbial organism, particularly apathogenic microbial organism; and/or 2) prevent onset of disease,reduce the risk of onset of disease, or reduce the severity of diseasesymptoms caused by infection by a microbial organism, particularly apathogenic microbial organism.

As used herein the term “isolated” is meant to describe a compound ofinterest that is in an environment different from that in which thecompound naturally occurs. “Isolated” is meant to include compounds thatare within samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

As used herein, the term “substantially purified” refers to a compoundthat is removed from its natural environment and is at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which it is naturally associated.

The term “artificial membrane” is meant to encompass a membrane thatprovides for incorporation of a functional PorB in the membrane (e.g., aPorB that can transport α-ketoglutarate), but which is not a part of aliving organism (e.g., a liposome, a lipid bilayer that is formed invitro and independent of a living cell, and the like).

By “subject” or “patient” or “individual” is meant any mammalian subjectfor whom diagnosis or therapy is desired, particularly humans. Othersubjects may include cattle, sheep (e.g., in detection of sheep at riskof abortion due to chlamydial infection), dogs, cats (e.g., in detectionof cats having eye and/or respiratory infections), birds (e.g., chickensor other poultry), guinea pigs, rabbits, rats, mice, horses, and so on.Of particular interest are subjects having or susceptible to Chlamydiainfection, particularly to infection by C. trachomatis, C. psittaciand/or C. pneumoniae.

The term “effective amount” or “therapeutically effective amount” meansa dosage sufficient to provide for treatment for the disease state beingtreated or to otherwise provide the desired effect (e.g., induction ofan effective immune response or reduction of bacterial load). Theprecise dosage will vary according to a variety of factors such assubject-dependent variables (e.g., age, immune system health, etc.), thedisease (e.g., the species of the infecting pathogen), and the treatmentbeing effected. In the case of an intracellular pathogen infection, an“effective amount” is that amount necessary to substantially improve thelikelihood of treating the infection, in particular that amount whichimproves the likelihood of successfully preventing infection oreliminating infection when it has occurred. Thus, for example, atherapeutic immune response is one that facilitates prevention ofinfection by a Chlamydial bacterium and/or facilitates clearance of aninfecting Chlamydia.

“Treatment” or “treating” as used herein means any therapeuticintervention in a subject, usually a mammalian subject, generally ahuman subject, including: (i) prevention, that is, causing the clinicalsymptoms not to develop, e.g., preventing infection and/or preventingprogression to a harmful state; (ii) inhibition, that is, arresting thedevelopment or further development of clinical symptoms, e.g.,mitigating or completely inhibiting an active (ongoing) infection sothat bacterial load is decreased to the degree that it is no longerseriously harmful, which decrease can include complete elimination of aninfectious dose of a Chlamydia bacteria from the subject; and/or (iii)relief, that is, causing the regression of clinical symptoms, e.g.,causing a relief of fever, inflammation, and/or other symptoms caused byan infection.

By the term “neutralizing epitope” as used herein is intended an aminoacid sequence that defines an antigenic determinant which is bound by anantibody and, in the context of infection, reduces infectivity of aChlamydial bacterium, e.g., by reducing the efficiency of bacterialinteraction with host cells important in establishing bacterialinfection or disease in the host, facilitating bacterial clearance, andthe like. “Neutralization” is intended to encompass any biologicalactivity of the bacteria, including reduction in the efficiency orability of the bacterium to establish infection or cause disease ordisease symptoms, inhibition of chlamydial EB formation, and the like.

As used herein, the term “neutralizing antibodies” refers to antibodieswhich bind a neutralizing epitope as described above.

By “neutralizing domain” is meant a sequence of contiguous amino acids,which sequence defines at least one neutralizing epitope.

Overview

The invention is based on the discovery of amino acid sequences of thechlamydial outer membrane porin protein, PorB, of C. trachomatis thatdefine neutralizing epitopes. The inventors have identified at leastfour domains of the PorB polypeptide which comprise at least oneneutralizing epitope. Polypeptides comprising at least one of thesedomains can be used to generate neutralizing antibodies againstChlamydia. In addition, the inventors have shown that the neutralizingepitopes of PorB elicit antibodies that are cross-reactive with PorB ofother chlamydial species. The PorB peptides of the invention thus areuseful in eliciting production of PorB-specific antibodies, whichantibodies can be used in, for example, detection or full-length PorBpolypeptide, purification of PorB, and can also serve as the basis forproduction of an effective vaccine

In one embodiment of particular interest, the Invention features PorBpolypeptides comprising neutralizing epitopes, as well as methods of useof such polypeptide to facilitate induction an immune response toneutralize Chlamydia infection.

PorB has several characteristics that make it an effective vaccine andchemotherapeutic target. Unlike other vaccine candidates such as MOMP,PorB does not vary substantially in its amino acid sequence betweenserovars and was instead highly conserved among the C. trachomatisstrains tested. This lack of variable regions indicates that PorB doesnot participate in antigenic variation that contributes to evasion ofthe immune response. PorB sequences between C. trachomatis and C.pneumoniae are also conserved further supporting a requirement forconstrained sequence to ensure its specific function, and providingfurther evidence that a vaccine based on a PorB polypeptide from onechlamydial species is cross-reactive with another chlamydial species,and thus can provide for induction of an immune response that canprovide immunoprotection across chlamydial species.

The invention thus provides compositions comprising a polypeptidecomprising at least one neutralizing epitope, and methods of inducinganti-chlamydial immunity based on these vaccines. In addition, theinvention also provides for detection of PorB polypeptides comprising atleast one neutralizing epitope or PorB-encoding sequences in diagnosisof Chlamydia infection.

The invention further features methods of identifying anti-chlamydialchemotherapeutics based upon identification of agents that inhibit PorBfunction in α-ketoglutarate transport.

Specific aspects of the invention will now be described in more detail.

PorB Peptide Compositions

In one aspect, the present invention provides compositions and methodsfor production of anti-chlamydial antibodies by administration of a PorBpeptide composition. In one embodiment, the compositions and methods ofthe invention are formulated as an immunogenic composition, andadministered to induce an immune response to infection by Chlamydia.Antigenic and immunogenic PorB peptides of the invention include, butare not necessarily limited to, peptides comprising a neutralizingepitope. Where the immunogenic composition comprises an antigenic PorBpeptide, the composition can further comprise any of a variety ofacceptable adjuvants, or the peptide can be coupled to a carrier.

“Polypeptides” and “peptides”, which are used interchangeably herein,are defined herein as organic compounds comprising two or more aminoacids covalently joined by an amide bond. Peptides may be referred towith respect to the number of constituent amino acids, e.g., a dipeptidecontains two amino acid residues, a tripeptide contains three, etc. Ingeneral, the peptides described herein do not encompass the full-lengthPorB polypeptide, and can be about 4, 6, 10 20, 30, 40, 50, 60 or moreamino acids in length, with peptides of from about 4 to about 47, fromabout 6 to about 34, and from about 6 to about 20 amino acids being ofparticular interest. In one embodiment, the PorB polypeptide comprisesat least one neutralizing epitope and is at least about 10 amino acidresidues, usually at least about 15 or 20 amino acid residues in length.

The peptides of the invention, while described herein as being composedof naturally occurring, L-amino acids, are not limited to such. Thepeptides described herein may be modified at the amino and/or carboxytermini; modified to contain the D-isomer rather than the normalL-isomer; modified chemically to have different substituents oradditional moieties; and the like, with the proviso that thesemodifications do not eliminate or otherwise adversely affect thepeptides ability to present a functional PorB epitope, particularly aneutralizing epitope of PorB. Exemplary chemical modifications of thepeptides include acylation, alkylation, esterification, amidification,etc. to produce structural analogs.

Furthermore, the peptides described herein can be modified by amino acidinsertion, deletion, addition, or substitution, again with the provisothat the modified peptide exhibits a PorB neutralizing epitope function.The amino acid substitutions may be of a conserved or non-conservednature. Conserved amino acid substitutions involve replacing one or moreamino acids of the peptide sequences described herein with amino acidsof similar charge, size, and/or hydrophobicity characteristics, such as,for example, a glutamic acid (E) to aspartic acid (D) amino acidsubstitution. Non-conserved substitutions involve replacing one or moreamino acids with amino acids possessing dissimilar charge, size, and/orhydrophobicity characteristics, such as, for example, a glutamic acid(E) to valine (V) substitution. Amino acid additions include additionsto the N-terminus, the C-terminus and/or a region between the N- andC-terminus. PorB peptides can also be provided as a fusion protein witha non-PorB amino acid sequence.

The peptides of the invention may be prepared by recombinant or chemicalsynthetic methods, which techniques are well known in the art. See, forexample, Creighton, 1983, Proteins: Structures and Molecular Principles,W. H. Freeman and Co., N.Y., which is incorporated herein by referencein its entirety. Short peptides, for example, can be synthesized on asolid support or in solution. Longer peptides may be made usingrecombinant DNA techniques. Here, the nucleotide sequences encoding thepeptides of the invention may be synthesized, and/or cloned, andexpressed according to techniques well known to those of ordinary skillin the art. See, for example, Sambrook, et al., 1989, Molecular Cloning,A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, N.Y.

When administered to a host, the PorB peptides of the inventioncomprising at least one neutralizing epitope and sufficient to elicit aprotective immune response) of PorB elicit an immune response (e.g., aprotective or therapeutic immune response). Polypeptides of theinvention include, but are not necessarily limited to, native PorBpolypeptide fragments (e.g., immunogenic, immunoprotective fragmentthereof, (e.g., a fragment of a PorB polypeptide comprising aneutralizing domain that, upon administration to a host, can elicit animmune response, preferably an immunoprotective immune response)), arecombinant form of a PorB peptide (e.g., a product of expression in aprokaryotic or eukaryotic recombinant host cell), a syntheticallyproduced PorB peptide, a modified recombinant or synthetic PorB peptide(e.g, PorB neutralizing epitope peptide provided as a fusion protein), aPorB peptide variant or analog that retains antigenicity orimmunogenicity of native PorB fragments having a neutralizing domain(e.g., an immunogenically similar or identical PorB-derived amino acidsequence), and the like. PorB peptides of interest are generally from atleast about 4 amino acids to about fragments of about 60 amino acids,usually at least about 6 amino acids, more usually at least about 10amino acids, and generally at least about 15 to 50 amino acids.Preferably, the PorB polypeptides have an amino acid sequence thatprovides for at least one neutralizing epitope domain. PorB peptides cancomprise an amino acid sequence of a PorB polypeptide of C. trachomatis,C. pneumoniae or C. psittaci, preferably of C. trachomatis.

A composition of interest comprises, in one embodiment, a PorB peptideof C. trachomatis, C. pneumoniae or C. psittaci, preferably a PorBpeptide of C. trachomatis. In one embodiment the C. trachomatispolypeptide comprises an amino acid sequence of an immunogenic fragmentfrom at least one of four PorB neutralizing epitope domains, ND1, ND2,ND3 and ND4. The composition can comprise any combination ofpolypeptides having at least one of these PorB neutralizing epitopedomains, and may comprise at least 2, 4, 6, 8, 9, 10 or more peptides.Generally at least two of the PorB neutralizing epitope domains arepresent in the composition, more usually at least three, and in someembodiments all four epitope domains are present. Table 1 provides theamino acid sequence for exemplary four neutralizing epitope domains ofPorB C. trachomatis, and also identifies the amino acid residues whichgenerally define each neutralizing domain.

TABLE 1 C. trachomatis PorB Neturalizing domains (ND) ND1 =FPVIPGINIEQKNACSFDLCNSYDVL Phe₃₄-Leu₅₉ (SEQ ID NO:7) ND2 =DLVNCNLNTNCVAVAFSLPDRSLSAIPLFDVSFE Asp₁₁₂-Glu₁₄ (SEQ ID NO:8) ND3 =GMIEVQSNYGFVWDVSLKKVIWKDGVSFVGVGADYR Gly₁₇₉-Ala₂₂₅ HASCPIDYIIA (SEQ IDNO:9) ND4 = VLPYLAFSIGSVSRQAPDDSFKKLEDRFTNLKFKVRK Val₂₆₁-Asn₃₀₅ ITSSHRGN(SEQ ID NO:10)

In certain embodiments, the immunogenic composition comprises multiplePorB peptides each having at least one different neutralizing epitopedomain. In one example, the composition comprises a PorB peptide havingan ND1 sequence or an antigenic or immunogenic portion thereof and apolypeptide having a ND2 sequence or an antigenic or immunogenic portionthereof. In other embodiments, the composition comprises peptides havingamino acid sequences, or antigenic or immunogenic portions thereof, ofeach of the four neutralizing epitope domains (ND1, ND2, ND3 and ND4) orany combination thereof.

Peptides having neutralizing domains of particular interest include, butare not necessarily limited to, peptides B1-2 (SEQ ID NO. 11), B1-3 (SEQID NO. 12) of neutralizing domain 1 (ND1); polypeptides B2-3 (SEQ ID NO.13), B2-4 (SEQ ID NO. 14) of ND2; polypeptides B3-2 (SEQ ID NO. 15),B3-3 (SEQ ID NO. 16), B3-4 (SEQ ID NO. 17) of ND3 and polypeptides B4-4(SEQ ID NO. 18), B5-1 (SEQ ID NO. 19) and B5-2 (SEQ ID NO. 20) of ND4.These exemplary peptides, as well as other peptides, are furtherdescribed in TABLE 2 and the Examples below.

In certain embodiments, the immunogenic composition of the inventioncomprises a combination of immunogenic or antigenic PorB polypeptides.For example, the composition can comprise a polypeptide of any of B1-2,B1-3, B2-3, B2-4, B3-2, B3-4 and B5-2 and any combination thereof. Forexample, the composition can comprise any combination of at least 2, 4,6, 8, 9, 10 or more of these exemplary polypeptides. Furthermore,polypeptides comprising overlapping amino acid residues betweenpolypeptides having SEQ ID NOs. 11-20 are also useful in thecompositions of the present invention.

A PorB polypeptide having a neutralizing epitope can be delivered to thehost in a variety of ways. For example, PorB peptides according to theinvention can be provided and administered as an isolated orsubstantially purified protein preparation. Alternatively or inaddition, the PorB peptides can be administered in the form of nucleicacid (e.g., RNA or DNA, usually DNA) encoding one or more, usually atleast 2, 4, 6, 8, 9, 10 or more, PorB peptides having at least oneneutralizing epitope (e.g., by genetic immunization techniques known inthe art), by delivery of shuttle vector (e.g., a viral vector (e.g., arecombinant adenoviral vector), or a recombinant bacterial vector (e.g.,a live, attenuated heterologous bacterial strain, e.g., live, attenuatedSalmonella) that provides for delivery of PorB polypeptide-encodingnucleic acid for expression in a host cell. Where nucleic acid encodinga PorB polypeptide is used in the immunogenic composition, the nucleicacid (e.g., DNA or RNA) can be operably linked to a promoter forexpression in a cell of the subject. Where two or more PorB peptides areadministered in the form of PorB-encoding nucleic acid, the PorBpeptides can be encoded on the same or different nucleic acid molecules.

Formulations

The PorB peptide compositions of the invention can be formulated in avariety of ways. In general, the compositions of the invention areformulated according to methods well known in the art using suitablepharmaceutical carrier(s) and/or vehicle(s). An exemplary suitablevehicle is sterile saline. Other aqueous and non-aqueous isotonicsterile injection solutions and aqueous and non-aqueous sterilesuspensions known to be pharmaceutically acceptable carriers and wellknown to those of skill in the art may be employed for this purpose.

Optionally, a composition of the invention may be formulated to containother components, including, e.g., adjuvants, stabilizers, pH adjusters,preservatives and the like. Such components are well known to those ofskill in the art.

The compositions can be administered in any suitable form that providesfor administration of the PorB peptides in an amount sufficient toelicit an immune response (e.g., humoral response, cellular response,and the like). For example, the composition can be administered as aliquid formulation or as a slow-release formulation (e.g., in a suitablesolid (e.g., biodegradable) or semi-solid (e.g., gel) matrix thatprovides for release of the PorB peptide or PorB peptide-encodingnucleic acid over time). The composition can be administered in a singlebolus, can be administered in incremental amounts over time, or anysuitable combination. In addition, it may be desirable to administer oneor more booster doses of the PorB peptides, which boosters may containthe same or different amounts of PorB peptide (or PorB peptide-encodingnucleic acid).

Administration of PorB Peptide Compositions

The PorB polypeptide immunogenic composition is administered in an“effective amount,” that is, an amount of PorB polypeptide or PorBpolypeptide-encoding nucleic acid that is effective in a route ofadministration to elicit a desired immune response, e.g., to elicitanti-PorB antibodies, e.g., to elicit anti-PorB antibody productionand/or to elicit an immune response effective to facilitate protectionof the host against infection by Chlamydia. For example, where PorBpolypeptide is delivered using a nucleic acid construct or a recombinantvirus, the nucleic acid construct or recombinant virus is administeredin an amount effective for expression of sufficient levels of theselected gene product to elicit production of anti-PorB antibodies,and/or to provide a vaccinal benefit, e.g., protective immunity.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, subcutaneous,transdermal, subdermal, intradermal, topical, rectal, oral and otherparental routes of administration. Routes of administration may becombined, if desired, or adjusted depending upon the immunogen or thedisease. As noted above, the PorB composition of the invention can beadministered in a single dose or in multiple doses, and may encompassadministration of booster doses, to elicit antibodies and/or maintainimmunity. Methods and devices for accomplishing delivery are well knownin the art. For example for administration through the skin, any of avariety of transdermal patches can be used to accomplish delivery.

The amount of PorB polypeptide, PorB polypeptide-encoding nucleic acid,or PorB polypeptide recombinant virions in each dose is selected as anamount which induces an immune response (particular an immunoprotectiveimmune response) without significant, adverse side effects. Such amountwill vary depending upon which specific immunogen is employed, whetheror not the immunogenic composition is adjuvanted, and a variety ofhost-dependent factors. Where PorB neutralizing epitope polypeptide isdelivered directly, it is expected that each does will comprise 1-1000μg of protein, generally from about 1-200 μg, normally from about 10-100μg. An effective dose of a PorB nucleic acid-based immunogeniccomposition will generally involve administration of from about 1-1000μg of nucleic acid. An optimal amount for a particular immunogeniccomposition can be ascertained by standard studies involving observationof antibody titres and other responses in subjects. Where theimmunogenic composition is administered as a prophylactic or therapeuticvaccine, the levels of immunity provided can be monitored to determinethe need, if any, for boosters. Following an assessment of antibodytiters in the serum, optional booster immunizations may be desired. Theimmune response to the protein of this invention is enhanced by the useof adjuvant and or an immunostimulant.

Subjects

Using the methods and compositions described herein in connection withthe subject invention, an immune response, including but not limited toan immunoprotective response, against chlamydial infection can beinduced in any subject, human or non-human. In one embodiment, thesubject to receive the PorB composition of the invention is one that issusceptible to infection by a chlamydial strain, particularly achlamydial strain pathogenic for the subject species. In general, themethods of the invention are effective to elicit an anti-chlamydialimmune response, with production of anti-PorB antibodies, particularlyantibodies that are cross-reactive with two or more chlamydial speciesbeing of particular interest. In one embodiment, the PorB composition ofthe invention is administered to as to facilitate prevention orinhibition of infection of the subject by a Chlamydia species thatexpresses on its surface a protein that is immunocrossreactive withPorB. In one embodiment administration of a PorB neutralizing epitopepolypeptide of C. trachomatis induces an immune response against C.trachomatis, C. pneumoniae and C. psittaci, particularly an immuneresponse against C. trachomatis. In another embodiment administration ofa PorB neutralizing epitope polypeptide of C. pneumoniae induces animmune response against C. trachomatis, C. pneumoniae and C. psittaci,and particularly an immune response against C. pneumoniae. In anotherembodiment, administration of a PorB neutralizing epitope polypeptide ofC. psittaci induces an immune response against infection by C.trachomatis, C. pneumoniae and C. psittaci, and particularly an immuneresponse against C. psittaci.

Human disease associated with chlamydial infection that can be mitigatedor prevented using the methods and compositions described hereininclude, but are not necessarily limited to, sexually transmitteddisease (urethritis and epidiymitis in men; pelvic inflammatory diseasein women), conjunctivitis, and pneumonia. Of particular interest is theinhibition or prevention of infection by C. trachomatis, by C.pneumonia, and by C. psittaci. Exemplary chlamydial diseases aredescribed in more detail below.

C. trachomatis, the most common cause of sexually transmitted diseasesin the United States, causes a variety of diseases includingnongonococcal urethritis and epididymitis in men; cervicitis,urethritis, and pelvic inflammatory disease in women; Reiter's syndrome;and neonatal conjunctivitis and pneumonia, the latter of which aregenerally acquired through maternal transmission. C. trachomatis hasbeen implicated in 20% of adults with pharyngitis. Several immunotypesof C. trachomatis can cause lymphogranuloma venereum (LGV), a diseasefound mostly in tropical and subtropical areas. LGV strains invade andreproduce in regional lymph nodes.

C. pneumoniae (previously called Taiwan acute respiratory agent orTWAR), originally considered a serotype of C. psittaci, can causepneumonia, especially in children and young adults. The organism hasbeen found in atheromatous lesions, and infection is associated withincreased risk of coronary artery disease.

C. psittaci infects many animals, but human infection is closely relatedto contact with birds. In humans, C. psittaci causes psittacosis, aninfectious atypical pneumonia transmitted to humans by certain birds. Inhumans, psittacosis (ornithosis, parrot fever) is usually caused byinhaling dust from feathers or excreta of infected birds or by beingbitten by an infected bird; rarely, it occurs by inhaling cough dropletsof infected patients or venereally. Human-to-human transmission may beassociated with highly virulent avian strains.

Where the subject is non-human, subjects of particular interest includerodent (e.g., mouse, rat, guinea pig, and the like), ungulate (e.g.,bovine, goat, sheep, and the like), feline, and avian subjects.

Anti-PorB Antibodies

Antibodies that specifically bind a PorB polypeptide having aneutralizing epitope can be used to isolate PorB polypeptides—eitherfull-length or partial peptides—or one or more chlamydial species.Anti-PorB antibodies can also be administered to provide temporary,passive immunity against chlamydial infections (e.g., to inhibitchlamydial infection and/or disease symptoms that can result from suchinfection). Methods for production of anti-PorB antibodies (e.g.,monoclonal or polyclonal antibodies) are well known in the art, as aremethods for formulating such antibodies for administration. In oneembodiment the anti-PorB antibody is a humanized antibody. Anti-PorBantibodies of interest also encompass modified antibodies (e.g.,modified to increase biological half-life following administration).

Methods for use of anti-PorB antibodies for isolation of PorBpolypeptides are well known in the art. For example, the anti-PorBantibody can be used to immunoprecipitate PorB peptides and polypeptidesin solution. Alternatively, the anti-PorB antibody can be bound to asolid support, such as an affinity column or a well of a microtiterplate, to facilitate immunopurification. Exemplary methods for PorBpolypeptide and peptide isolation and/or purification are provided belowin the context of diagnostic assays, and can be readily adapted toincrease the yield of PorB

In therapeutic applications, anti-PorB antibodies are administered in anamount sufficient to neutralize Chlamydia so as to prevent, mitigate, orreduce the likelihood of onset of infection. Anti-PorB antibodies can beadministered by any suitable route, generally by parenteral injection(e.g., subcutaneous, intramuscular, intravenous, etc.). Administrationof anti-PorB antibodies particularly useful for preventing or inhibitinginfection in immunocompromised subjects or other subjects having animmune system that can not maintain an effective, immunoprotectiveresponse to, for example, a PorB antigen.

Antibodies that bind PorB of two or more Chlamydial species are ofparticular interest, particularly antibodies that bind-PorB epitopes ina manner effective to block infection by each of these species are ofparticular interest. For example, antibodies that bind PorB of C.trachomatis as well as PorB of C. pneumoniae and/or C. psittaci, can beused to provide passive immunity that protects against infection by eachof these chlamydial species.

Diagnosis of Chlamydial Infection

In addition to the uses described above, PorB peptides having aneutralizing epitope and sequences obtained from PorB-encodingpolynucleotides can be used in the detection of Chlamydia infection in asubject and/or determining whether a subject has been exposed toChlamydia infection. Diagnostic assays based upon detection of PorB or aPorB-encoding sequence in a biological sample include, but are notnecessarily limited to, detection of PorB peptides, detection ofanti-PorB antibodies, and/or detection of PorB peptide-encodingsequences in a test sample from the subject. Detection of any of thesePorB peptide, polynucleotides, or antibodies in a sample taken from asubject is indicative of chlamydial infection in the subject. Forclarity, applicants note that “probe” as used herein in the context ofdetection of PorB polypeptides or PorB polypeptide-encodingpolynucleotides is meant to encompass anti-PorB antibodies (e.g., fordetection of PorB polypeptide), PorB polypeptide or PorB peptides (e.g.,for detection of anti-PorB antibodies), and PorB polynucleotides andfragments thereof (e.g., for use in hybridization or PCR assays todetect PorB polynucleotides).

In one general embodiment, the methods of the invention involvesdetecting PorB peptides or anti-PorB antibodies in the subject bycontacting an appropriate biological test sample from a subjectsuspected having been exposed to Chlamydia or suspected of having aChlamydia infection with a probe that is one of a) an antibody thatspecifically binds a PorB polypeptide, b)a PorB polypeptide. After thetest sample is contacted with the probe for a time sufficient to allowfor formation of specific antibody-PorB polypeptide binding pairs, theformation of such binding pairs is detected. Detection of theantibody-PorB binding pairs indicates that the subject has been exposedto Chlamydia (due to the presence of PorB polypeptides in the samplewhere the probe is an anti-PorB antibody), or has mounted an immuneresponse to PorB polypeptide (due to the presence of anti-PorBantibodies in the sample, as detected using a PorB polypeptide probe)which suggests at least prior exposure to Chlamydia. In one embodimentof particular interest, the probe is a PorB peptide comprising at leastone neutralizing domain, which is used to detect to isolate anti-PorBantibodies.

In another general embodiment, the presence of a Chlamydia infection inthe host is detected using an probe to specifically hybridizes to and/orspecifically amplifies (e.g., through use of PCR) to a PorB polypeptideencoding polynucleotide. Detection of hybridization or detection of aspecific PCR product indicates that the subject has a Chlamydiainfection.

Diagnosis Based on Detection of PorB Polypeptides and/or Anti-PorBAntibodies

Detection of PorB polypeptides can be accomplished according to a widevariety of immunoassays that are well known in the art, and may beperformed either qualitatively or quantitatively. For example, thediagnostic assay can measure the reactivity between an anti-PorBantibody (e.g., a polyclonal or monoclonal antibody (MAb), preferably aMAb) and a patient sample, usually a sample of a bodily fluid, e.g.,mucosal secretion, blood-derived sample (e.g., plasma or serum), urine,and the like. The patient sample may be used directly, or diluted asappropriate, usually about 1:10 and usually not more than about1:10,000. Immunoassays may be performed in any physiological buffer,e.g. PBS, normal saline, HBSS, PBS, etc.

In one embodiment, a conventional sandwich type assay is used. Asandwich assay is performed by first immobilizing proteins from the testsample on an insoluble surface or support. The test sample may be boundto the surface by any convenient means, depending upon the nature of thesurface, either directly or indirectly. The particular manner of bindingis not crucial so long as it is compatible with the reagents and overallmethods of the invention. They may be bound to the plates covalently ornon-covalently, preferably non-covalently.

The insoluble supports may be any compositions to which the test samplepolypeptides can be bound, which is readily separated from solublematerial, and which is otherwise compatible with the overall method ofdetecting and/or measuring type I cell- or type II cell-specificpolypeptide. The surface of such supports may be solid or porous and ofany convenient shape. Examples of suitable insoluble supports to whichthe receptor is bound include beads, e.g. magnetic beads, membranes andmicrotiter plates. These are typically made of glass, plastic (e.g.polystyrene), polysaccharides, nylon or nitrocellulose. Microtiterplates are especially convenient because a large number of assays can becarried out simultaneously, using small amounts of reagents and samples.

Before adding patient samples or fractions thereof, the non-specificbinding sites on the insoluble support, i.e. those not occupied bypolypeptide, are generally blocked. Preferred blocking agents includenon-interfering proteins such as bovine serum albumin, casein, gelatin,and the like. Alternatively, several detergents at non-interferingconcentrations, such as Tween, NP40, TX100, and the like may be used.

Samples, fractions or aliquots thereof can be added to separatelyassayable supports (for example, separate wells of a microtiter plate).A series of standards, containing known concentrations of PorB can beassayed in parallel with the samples or aliquots thereof to serve ascontrols and to provide a means for quantitating the amounts of PorBpolypeptide present in the test sample. Preferably, each sample andstandard will be added to multiple wells so that mean values can beobtained for each.

After the test sample polypeptides are immobilized on the solid support,anti-PorB antibody is added. The incubation time of the sample and theantibody should be for at time sufficient for antibody binding to theinsoluble polypeptide. Generally, from about 0.1 to 3 hr is sufficient,usually 1 hr sufficing.

After incubation, the insoluble support is generally washed of non-boundcomponents. Generally, a dilute non-ionic detergent medium at anappropriate pH, generally 7-8, is used as a wash medium. From one to sixwashes may be employed, with sufficient volume to thoroughly washnon-specifically bound proteins present in the sample. After washing,antibody binding to the sample can be detected by virtue of a detectablelabel on the antibody. Where the antibody is not detectably labeled,antibody binding can be detected by contacting the sample with asolution containing antibody-specific second receptor, in most cases asecondary antibody (i.e., an anti-antibody). The second receptor may beany compound which binds antibodies with sufficient specificity suchthat the bound antibody is distinguished from other components present.In a preferred embodiment, second receptors are antibodies specific forthe anti-PorB antibody, and may be either monoclonal or polyclonal sera,e.g. goat anti-mouse antibody, rabbit anti-mouse antibody, etc.

The antibody-specific second receptors are preferably labeled tofacilitate direct, or indirect quantification of binding. Examples oflabels which permit direct measurement of second receptor bindinginclude light-detectable labels, radiolabels (such as ³H or ¹²⁵I),fluorescers, dyes, beads, chemiluminescers, colloidal particles, and thelike. Examples of labels which permit indirect measurement of bindinginclude enzymes where the substrate may provide for a colored orfluorescent product. In a preferred embodiment, the second receptors areantibodies labeled with a covalently bound enzyme capable of providing adetectable product signal after addition of suitable substrate. Examplesof suitable enzymes for use in conjugates include horseradishperoxidase, alkaline phosphatase, malate dehydrogenase and the like.Where not commercially available, such antibody-enzyme conjugates arereadily produced by techniques known to those skilled in the art.

Alternatively, the second receptor may be unlabeled. In this case, alabeled second receptor-specific compound is employed which binds to thebound second receptor. Such a second receptor-specific compound can belabeled in any of the above manners. It is possible to select suchcompounds such that multiple compounds bind each molecule of boundsecond receptor. Examples of second receptor/second receptor-specificmolecule pairs include antibody/anti-antibody and avidin (orstreptavidin)/biotin. Since the resultant signal is thus amplified, thistechnique may be advantageous where only a small amount of PorBpolypeptide is present, or where the background measurement (e.g.,non-specific binding) is unacceptably high. An example is the use of alabeled antibody specific to the second receptor. More specifically,where the second receptor is a rabbit anti-allotypic antibody, anantibody directed against the constant region of rabbit antibodiesprovides a suitable second receptor specific molecule. The anti-Ig willusually come from any source other than human, such as ovine, rodentia,particularly mouse, or bovine.

The volume, composition and concentration of anti-antibody solutionprovides for measurable binding to the antibody already bound toreceptor. The concentration will generally be sufficient to saturate allantibody potentially bound to PorB polypeptide. The solution containingthe second receptor is generally buffered in the range of about pH6.5-9.5. The solution may also contain an innocuous protein aspreviously described. The incubation time should be sufficient for thelabeled ligand to bind available molecules. Generally, from about 0.1 to3 hr is sufficient, usually 1 hr sufficing.

After the second receptor or second receptor-conjugate has bound, theinsoluble support is generally again washed free of non-specificallybound second receptor, essentially as described for prior washes. Afternon-specifically bound material has been cleared, the signal produced bythe bound conjugate is detected by conventional means. Where an enzymeconjugate is used, an appropriate enzyme substrate is provided so adetectable product is formed. More specifically, where a peroxidase isthe selected enzyme conjugate, a preferred substrate combination is H₂O₂and is O-phenylenediamine which yields a colored product underappropriate reaction conditions. Appropriate substrates for other enzymeconjugates such as those disclosed above are known to those skilled inthe art. Suitable reaction conditions as well as means for detecting thevarious useful conjugates or their products are also known to thoseskilled in the art. For the product of the substrate O-phenylenediaminefor example, light absorbance at 490-495 nm is conveniently measuredwith a spectrophotometer.

The absence or presence of antibody binding may be determined by variousmethods that are compatible with the detectable label used, e.g.,microscopy, radiography, scintillation counting, etc. Generally theamount of bound anti-PorB antibody detected will be compared to controlsamples (e.g., positive controls containing PorB or negative controlslacking such polypeptides). The presence of anti-PorB antibody isindicative of the presence of a Chlamydia in the test sample, which inturn is indicative of chlamydial infection in the subject.

As will be readily appreciated by the ordinarily skilled artisan uponreading the present disclosure, the above techniques can be readilymodified to provide for detection of anti-PorB antibodies in the host.For example, rather than immobilizing PorB polypeptide on a solidsupport, an anti-PorB antibody is immobilized on the support andsubsequently contacted with a test sample from the host. Binding of PorBpolypeptide from the test sample to the support-bound anti-PorB antibodycan then be detected using a second anti-PorB antibody (e.g., that bindsto a different epitope of the polypeptide than the bound antibody).Binding of the second antibody can then be detected according to methodswell known in the art.

Diagnosis Based on Detection of PorB Nucleic Acid

Where the diagnostic assay involves detection of a PorB-encodingsequence, the assay can take advantage of any of a variety ofpolynucleotide detection techniques that are well known in the art. Forexample, a fragment of a PorB-encoding sequence can be used as a probeto detect hybridizing sequences in a test sample, or for use as a primerin PCR amplification of chlamydial nucleic acid in at test sample.Methods for detecting sequences based on hybridization, as well as useof PCR are known in the art, see, e.g., Sambrook, et al. MolecularCloning: A Laboratory Manual, CSH Press 1989. The probe or primer maycomprise a detectable label. Suitable labels include fluorochromes, e.g.fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin,allophycocyanin, 6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the polynucleotide is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. In PCR,the label may be conjugated to one or both of the primers;alternatively, the pool of nucleotides used in the amplification islabeled, so as to incorporate the label into the amplification product.

Kits for Detection of Chlamydia

PorB peptides (for detection of specific antibodies), anti-PorBantibodies, and PorB peptide-encoding polynucleotide probes and/orprimers, as well as other materials useful in the diagnostic methods ofthe invention (e.g., labels, compounds for detection of labels, solidsupports for capture of nucleic acid in a sample, filters for at leastpartial separation or purification of parasites in the sample,detergents and other reagents (e.g., lysing mammalian and cells in thesample), etc.) can be provided in a kit. Such kits can include samplesto serve as positive controls or negative controls. Preferably such kitsare designed for use in the field, e.g., do not contain components thatrequire refrigeration, are portable, etc.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Methods and Materials for Examples 1-12

The following procedures are used in the Examples described in detailbelow. Although some of the methods described below are in common use,the specific protocol used in the Examples below is described in detailwhere alternative protocols are often employed. Basic procedures such asDNA digestion by restriction enzymes and ligation are not described, assuch are well within the skill of the ordinarily skilled artisan and, insome instances, are carried out according to the enzyme or kitmanufacturer's instructions.

Chlamydial cultures. C. trachomatis strains B/TW-5/OT, C/TW-3/OT, andL2/434/Bu were grown in L929 cells, and strain D/UW-3/Cx was grown inHeLa 229 cells. Elementary bodies (EB) and reticulate bodies (RB) wereseparately purified by diatrizoate (Renograffin; E. R. Squibb and Sons,Princeton, N.J.) gradients and were used immediately after purificationor stored at −70 C. RB was purified at 24 hours post-infection.

Bacterial strains and plasmid. The synthetic gene encoding MOMP (ompA)was constructed in E. coli HMS 174 (DE3) and has been previouslydescribed (Jones et al. (2000) Gene 258:173-181). E. coli HMS 174 (DE3)without the plasmid was used as a control strain. PorB cloning andexpression were done in the E. coli strain, TOP10 (Invitrogen, Carlsbad,Calif.). The complete PorB gene was cloned into the pBAD-TOPO TA vector(Invitrogen), which contains the araBAD promoter.

Expression and purification of PorB. Regulation of expression is by theAraC gene product on the promoter in the absence or presence ofarabinose. All E. coli cultures were grown with aeration at 37° C. inLuria-Bertani broth containing 100 mg/ml of ampicillin until thecultures reached an O.D of 0.6. 0.02% Arabinose to a final concentrationof 0.5 mM was added to induce the expression of PorB. PorB was clonedwith a C-terminal HIS tag and purified by nickel column using the HISBind Purification system (Novagen, Madison, Wis.). Extraction of PorBwith 1% octylglucoside at 37° C. for 1 h and dialysis of the detergentout of the extracted PorB using PBS and then 1× Bind buffer (Novagen)was necessary before purification by nickel column. IPTG was added to afinal concentration of 0.5 mM to induce the expression of MOMP. Theouter membranes of E. coli expressing MOMP were purified as described inMOMP Jones et al. (2000 Gene 258:173-181)

Outer membrane preparation. The spheroplasts and outer membranes of E.coli were isolated using the method of Osborn and Munson (1974 MethodsEnzymol. 31:642-653) with the following modifications. The E. coli weregrown in Luria Bertani broth with 100 mg/ml ampicillin at 37° C. withvigorous aeration to a density of approximately 5×10⁸ bacteria/ml,followed by 2 hours of induction by addition of 0.02% arabinose to aconcentration of 0.5 mM. 25 ml aliquots of the spheroplasts were lysedby sonication by immersing in an ice-salt bath and sonicating for three15-second periods with a Braunsonic U sonicator. The suspension wascooled for 1 minute between bursts. The unbroken cells were removed bycentrifugation at 1200×g for 15 minutes at 4° C. The supernatantfraction was then centrifuged for 2 hours at 100,000×g at 4° C. Themembrane pellet was resuspended in a small volume of cold 0.25 Msucrose-3.3 mM Tris-1 mM EDTA, pH 7.8 and centrifuged for 2 hours at100,000×g 4° C. The pellet was then suspended in 6 ml of cold 25%sucrose −5 mM EDTA, pH 7.5 for separation by isopycnic centrifugation.An outer membrane preparation was performed with a control cloneexpressing a non-outer membrane protein and this protein was notdetected in the outer membrane fraction.

Chlamydial outer membrane complex (COMC) preparation. The COMC wasprepared from fresh, not previously frozen, purified EB (10 mg) andperformed according to the method of Caldwell et al. (Caldwell et al.(1981) Infect. Immun. 31:1161-1176) with some modifications. EB weresuspended in 3 ml of 10 mM sodium phosphate buffer (pH 7.4) and 2%SARKOSYL. This suspension was sonicated briefly and centrifuged at100,000×g for 1 hour at 20° C. Both the soluble and insoluble (COMC)fractions were analyzed by SDS-PAGE.

Antibodies. Polyvalent monospecific antisera to PorB were obtained frommice Swiss-Webster mice immunized with 1) nickel column-purified PorBprotein and 2) a piece of PorB consisting of the amino-terminal portion,from amino acid 24-71 (PorB²⁴⁻⁷¹). The mice were immunized twice attwo-week intervals with 100 μg of purified protein in an equal volume ofcomplete Freund's adjuvant for the second immunization. IH5 is a L2serovar specific monoclonal antibody specific to MOMP. Polyvalentantiserum produced in rabbits using L2 EB and polyvalent monospecificantiserum produced in rabbits using cloned and expressed 28 kDa plasmidprotein (pgp3) (Comanducci et al. (1993) J. Gen. Microbiol.139:1083-1092) were used in the dot blot experiment.

Cell staining. C. trachomatis serovar L2-infected, D-infected anduninfected HeLa cells were fixed in methanol for 10 minutes and washedthree times in PBS. The anti-PorB monospecific antibody was diluted1:200, added to the cells and incubated for 1 hour at room temperatureon a rocker platform. The monolayer was rinsed three times in PBS andoverlaid with a fluorescein isothiocyanate-conjugated anti-mouseimmunoglobulin G (Zymed, So. San Francisco, Calif.) diluted 1:50. Thecells were incubated in the dark for one hour at room temperature on arocker platform and then washed three times with PBS. The cells werethen counter stained with Evans blue and observed by fluorescencemicroscopy.

Dot Blot assay. Dot blots were performed as previously described byZhang et al., (1987 J. Immunol. 138:575-581) with the followingdifferences: 1) the method of detection was enhanced chemiluminescence(ECL) (Amersham Pharmacia Biotech, Piscataway, N.J.); 2) an anti-mouseHRP-conjugated secondary antibody was used; 3) the primary and secondaryantibodies were washed by rinsing the wells with PBS and discarding thePBS. Vacuum filtration was used after the final wash to remove allliquid from the wells.

Dot blots of viable chlamydial EB to determine surface accessibility ofPorB were performed by probing immobilized EB with (1) a negativecontrol antibody, anti-pgp3; (2) a positive control monoclonal, IH5; (3)an anti-PorB antibody; (4) an anti-PorB²⁴⁻⁷¹ antibody; and (5) apositive control polyclonal, anti-L2 EB was used. The anti-pgp3 antibodywas used at 1:1000 for the immunoblot and bound a 28 kDa protein, whileit was used at 1:100 for the dot blot. The rabbit anti-L2 EB polyclonalantibody was used at 1:1000 for both the immunoblot and dot blot. TheIH5 monoclonal antibody was used at 1:1000 for the immunoblot and at1:4000 for the dot blot. The anti-PorB antibody was used at 1:200 forthe immunoblot and at 1:100 for the dot blot.

Protease cleavage. Fresh EB, not previously frozen, were incubated withvarious concentrations of trypsin (0, 0.001, 0.01, 0.1 mg/ml) andproteinase K (0, 0.1, 0.5, 1 mg/ml) for 30 minutes at 37° C. The treatedEB were then immediately transferred to a nitrocellulose membrane and adot blot analysis was performed as describe above.

Neutralization assay. The HaK (hamster kidney cells) in vitroneutralization assay was performed as previously described (Byrne et al.(1993) J. Infect. Dis. 168:415-20). Antibodies, except for pre-immuneserum, were quantitated and diluted to 200 mg/ml, then serially dilutedby two-fold to 12.5 mg/ml. Pre-immune serum was used at a dilution of1:10 and serially diluted 2-fold to 1:160. For detection of PorB,monospecific anti-PorB was purified with protein A (Sigma, St. Louis,Mo.), filter sterilized, quantitated using the BCA assay (Pierce,Rockford, Ill.), and diluted in SPG to the appropriate concentrations. Acontrol monoclonal antibody with specificity for MOMP (IH5) was used.Also, a control monoclonal antibody with unrelated specificity, theanti-pgp3 antibody as well as the pre-immune serum were used ascontrols. L2 EB was diluted in SPG to contain 2×10⁴ IFU/ml, 100 ml wasadded to each antibody dilution in total volume of 200 ml.Neutralization proceeded for 30 minutes. IFU were quantitated bycounting ten fields at a magnification of 40×. A mean IFU per field wascalculated and the results were shown as percent reduction in mean IFUcompared with the control plates.

Quantitation of protein. Purified protein and outer membranes for use inthe liposome swelling assay was quantitated according to the Lowrymethod. All other samples were quantitated by the BCA assay (Pierce,Rockford, Ill.).

Liposome swelling assay. The liposome swelling assay was performedaccording to the method of Nikaido (Nikaido & Rosenberg (1983) J.Bacteriol. 153:241-252) with the following modifications: 1) liposomeswere made by mixing 5.0 μmol phosphatidylcholine and 0.02 μmoldicetylphosphate with outer membrane proteins or purified protein inorder to increase the optical density readings to the range of 0.4-0.7O.D., and 2) the liposome drying time was longer than 2 minutes (i.e., 5minutes), but at a lower temperature of 37° C. Liposomes were made witheither dextran T-40 (15% dextran T-40 in 5 mM Tris-Cl, pH7.5) orstachyose inside. Since stachyose is impermeable to the porins, it wasused as a control to determine the isoosmotic concentration of othersolutes. The concentration of stachyose which produced no swelling orshrinking of the proteoliposomes was determined to be the isoosmoticconcentration. The swelling rates were determined as d(1/OD)/dt from theoptical density changes between 10 and 20 seconds (Nikaido & Rosenberg(1983) J. Bacteriol. 153:241-252).

Liposome swelling assay for testing anions. Liposomes were madeaccording to the method described above with a few modifications. Thefollowing was added to phosphatidylcholine and dicetylphosphate driedwith PorB (6 μg): 4 mM NAD⁺, 12 mM stachyose, 1 mM imidazole-NAD bufferpH (6.0). The test solution consisted of 1 mM Imidazole-NAD (pH 6.0), 1mM Sodium NAD, 6 mM disodium salt of the anion to be tested(α-ketoglutarate, succinate, oxaloacetate, malate, or citrate). Controlliposomes without protein were used to determine the isotonicconcentration of the test solutions.

Enzyme-linked liposome swelling assay. Liposomes were made as describedabove with addition of 50 mM potassium phosphate, 2.5 mM NAD⁺, 0.2 mMthiamin pyrophosphate, 1.0 mM magnesium chloride, 0.13 mM coenzyme A,2.6 mM cysteine, and 5.0 units of α-ketoglutarate dehydrogenase. Variousconcentrations of α-ketoglutarate (0.001 mM-1 mM) were used as testsolutes. Liposomes containing PorB (6 μg) and control liposome withoutprotein were made with the reaction mixture, washed through a SEPHADEXcolumn (S-300) equilibrated with reaction mixture withoutα-ketoglutarate dehydrogenase, and placed inside a cuvette.α-ketoglutarate was added to the reaction and mixed. The formation ofNADH was measured by the increase in O.D.₃₄₀.

Example 1 Analysis of PorB Sequence—Comparison to Major Outer MembraneProtein (MOMP)

Genome sequence analysis revealed a number of predicted outer membraneproteins (see Stephens et al. 1998 “Genome sequence of an obligateintracellular pathogen of humans: Chlamydia trachomatis” Science282:754-759). One such predicted outer membrane protein, encoded by thepredicted open reading frame CT713, was selected for analysis, andreferred to herein as PorB. The nucleotide and amino acid sequences ofPorB (CT713) are available within the complete sequence of the genome atGenBank Accession No. NC_(—)000117, with the amino acid sequence atGenBank Accession No. gi|3329169. The open reading frame correspondingto PorB is the complement of nucleotide residues 3616 to 4638 of GenBankAccession No. AE001342. The nucleotide and amino acid sequences of PorBof C. trachomatis are provided in the Sequence Listing as SEQ ID NOS:1and 2, respectively. Alignment of the amino acid sequence of PorB withthe amino acid sequence of MOMP is provided in FIG. 1.

As illustrated in FIG. 1, PorB has only slight sequence similarity(20.4%) to MOMP. Despite this relatively low amino acid sequencesimilarity, PorB and MOMP do share certain characteristics andstructural features. The estimated size of this protein is 38,000daltons and the isoelectric point was calculated to be 4.9. MOMP has amolecular weight of 40,000 with an isoelectric point calculated andexperimentally confirmed to be 5.0 (Bavoil et al. (1984) Infect. Immun.44:479-485). PorB has a predicted cleavable leader sequence as well asan amino acid sequence which ends in phenylalanine (arrow in FIG. 2), acharacteristic of many outer membrane protein (Struyvé et al. (1991) J.Mol. Biol. 218:141-148). Both PorB and MOMP have the same number ofcysteines (9 cysteines) suggesting that PorB may be an outer membranecysteine-rich protein analogous to, although distinct from, MOMP.

Previous reports on outer membrane proteins of Chlamydia have notidentified this protein. The overabundance of MOMP and similarity insize and isoelectric point likely contributed in preventing earlierdetection of PorB. PorB is not as predominant as MOMP by approximately20-fold. Since PorB is similar in size to MOMP, an SDS-PAGE analysis ofchlamydial outer membrane complexes can not distinguish PorB from MOMP.Also, PorB has a similar isoelectric point to MOMP, therefore a 2-D gelanalysis may not separate the proteins (Bavoil et al. (1984) Infect.Immun. 44:479-485; Bini et al. (1996) Electrophoresis 17:185-190).

Example 2 Analysis of PorB Sequence—Comparison of PorB Amino AcidSequences from Different Serovars

When compared with other serovars of C. trachomatis, MOMP has fourdistinct variable segments which correspond to surface exposed regionsof the protein. Serovar designations have been related to thedifferences in these variable segments of MOMP (Stephens et al. (1988)J. Exp. Med. 167:817-831). In order to determine whether this serovarvariation is also characteristic for PorB, the sequence of PorB betweenserovars was compared.

FIG. 2 provides an alignment of the amino acid sequences of PorB fromthe C. trachomatis serovars D (CT-D) (SEQ ID NO:2), L2 (CT-L2) (SEQ IDNO:5), and C (CT-C) (SEQ ID NO:6), as well as the amino acid sequence ofPorB from C. pneumoniae (CPn) (SEQ ID NO:4). The PorB of C. trachomatisand C. pneumoniae are 59.4% identical. C. trachomatis serovar L2 and Cdifferences are indicated below the amino acid sequence. The cysteinesare indicated with an asterisk above the amino acid sequence.

The nucleotide and amino acid sequence alignments between serovars D, B,C and L2 revealed no to only minor differences. There is no PorBsequence difference between serovars D and B, while there is onenucleotide difference, which results in an amino acid change, betweenserovars D (or B) and C. Between serovars D (or B) and L2 there are sixnucleotide differences, each of which result in a difference in theencoded amino acid. The nucleotide differences occur throughout the geneand were not clustered to any region (FIG. 2). Among the serovarsinvestigated, there are no variable segments in PorB such as there arein MOMP. Thus, sequence variation is not a phenotype for PorB.

Comparison between PorB of C. trachomatis (serovar D) and C. pneumoniaereveals greater differences dispersed throughout the gene. However, with59.4% identity between amino acid sequences of C. trachomatis and C.pneumoniae, this protein is highly conserved between species (FIG. 2).C. pneumoniae PorB has 6 cysteines, four of which are conserved betweenspecies, while C. trachomatis serovars D, B and C have 9 conservedcysteines and serovar L2 has 8.

Example 3 Expression of PorB in E. coli

PorB was predicted to be in the outer membrane through a variety ofprotein localization programs such as PSORT (K. Nakai, Human GenomeCenter, Institute for Medical Science, University of Tokyo, Japan). Aleader sequence cleavage site for C. trachomatis PorB was predicted tobe at amino acid 26 (FIG. 2). The complete gene including the leadersequence was cloned into E. coli with a HIS tag at the C-terminal end ofPorB and expressed. The protein was affinity purified by nickel columnchromatography.

PorB expressed in E. coli was localized to the outer membrane fractionas determined by an immunoblot using an antibody to the C-terminal HIStag. E. coli porins were also detected in this outer membrane fractionby Coomassie stain. The presence of PorB was primarily localized to theouter membrane suggesting that PorB has the necessary signal(s) to betransported to the outer membrane by E. coli.

Example 4 Presence of PorB in Inclusions

In order to characterize PorB in Chlamydia, a polyclonal monospecificserum was produced to the complete purified protein. FITC cell stainingexperiments using the anti-PorB serum showed that this serum containedantibody that bound antigens localized to the inclusions in infectedcells. Anti-PorB serum did not label uninfected control cells. Stainingcells infected with serovar L2 and serovar D, 48 and 72 hours postinfection, respectively with anti-PorB serum revealed punctate stainingconsistent with the morphology for EB and RB. This antibody staining waspresent at 10, 15, 20, 24, 48 hours post infection, indicating that thisprotein is constitutively expressed and/or present throughout thechlamydial development cycle.

Example 5 Localization of PorB to the COMC

The anti-PorB antibody bound a protein in Chlamydia that was similar insize to MOMP by immunoblot analysis. The amount of PorB present in EBand RB was similar. The serum also bound the purified HIS-taggedprotein, which was detected by an anti-HIS antibody. Although there wereonly slight similarities in sequence to MOMP, testing for crossreactivity between antibodies to PorB and MOMP was performed. Anti-PorBserum did not bind MOMP expressed in E. coli. Therefore, it is concludedthat the anti-PorB sera bound PorB and did not cross react with MOMP.

In order to determine if PorB is a component of the Chlamydia OuterMembrane Complex (COMC), the COMC was isolated and probed with anti-PorBserum. Since the chlamydial outer membrane is highly disulfide bonded,the Sarkosyl insoluble fraction contains a number of proteins such asMOMP and other cysteine rich proteins. PorB was detected in the COMCfraction and not the soluble supernatant. Therefore, the presence ofPorB in the COMC fraction demonstrates that this protein is in thechlamydial outer membrane and is disulfide linked perhaps to other COMCproteins.

Example 6 Surface Accessibility of PorB

Since PorB was predicted to be in the outer membrane and was localizedto the COMC, surface accessibility of this protein was tested. Dot blotexperiments have been shown to be specific for surface accessibleantigens (Zhang et al. (1987) J. Immunol. 138:575-581) and was used totest surface accessibility of PorB. The dot blot using the anti-PorBsera showed that this antibody bound EB. A negative control rabbitpolyclonal serum to a 28 kDa plasmid protein (pgp3) was used as anegative control antibody since this protein is not present in the outermembrane of Chlamydia (Comanducci et al. (1993) J. Gen. Microbiol.139:1083-1092). This negative control antibody did not bind EB, while apositive control antibody to a surface accessible antigen on MOMP (IH5)bound. These data demonstrate that PorB is localized to the outermembrane.

Example 7 Effect of Proteolytic Cleavage on PorB

To investigate surface exposure of PorB, purified EB were digested withproteases and proteins from EB were assessed for binding by theanti-PorB antibody. Using the dot blot method, EB were treated withvarious concentrations of trypsin or proteinase K, immobilized on anitrocellulose membrane and probed with the anti-PorB antibody, as wellas to antibodies to MOMP and the anti-pgp3 antibody. A reduction inbinding by anti-PorB antibodies was observed for EB-digested proteinssuggesting that PorB has surface accessible trypsin and proteinase Kcleavage sites, and thus is an outer membrane protein.

Example 8 Neutralization of C. trachomatis By Anti-PorB

Since PorB is an outer membrane protein with surface exposed regions,antibodies made to PorB were tested for ability neutralize infectivityof C. trachomatis (serovar L2). The anti-PorB sera produced using eitherthe entire protein or an amino-terminal fragment (amino acids 24-71) ata concentration of 100 mg/ml neutralized infectivity by up to 88% and70%, respectively, further supporting the conclusion that PorB is asurface exposed outer membrane protein (FIG. 3). The control antibodywithout specificity to outer membrane proteins, anti-pgp3, as well asthe pre-immune sera did not neutralize infectivity (FIG. 3). Amonoclonal antibody to serovar L2 MOMP (IH5) at a concentration of 50and 100 mg/ml neutralized infectivity up to 78% (FIG. 3). Thisneutralization assay confirms that antibodies to PorB can inhibitinfectivity by C. trachomatis since this assay is an art-recognized invitro correlate for the assessment of protective immunity (Byrne et al.(1993) J. Infect. Dis. 168:415-20).

Example 9 Pore-Forming Activity of PorB

The pore-forming capabilities of PorB were tested using the liposomereconstitution assay (Nikaido (1983) Methods Enzymol. 97:85-95). Theliposome swelling assay for study of porin function is used not onlybecause it is well established, but because this assay gives preciseinformation on the rates of diffusion of solutes through the porinchannels (Nikaido & Rosenberg (1983) J. Bacteriol. 153:241-252). Thisassay involves the formation of liposomes incorporated with pore-formingprotein and then determination of whether and how fast test solutes candiffuse through the protein channels. This assay was used to test andcompare pore-forming activity of the C. trachomatis PorB and MOMP.

Purification of MOMP using mild detergents causes a loss in porinactivity (Bavoil, et al. (1984) Infect. Immun. 44:479-485, Wyllie, etal. (1998) Infect. Immun. 66:5202-5207), therefore, MOMP was expressedin E. coli and outer membrane fractions enriched for MOMP were used. Ithas been shown in liposome swelling assays that the predominant porinactivity of the outer membrane fraction of E. coli expressing MOMP isdue to MOMP (Jones et al. (2000) Gene 258:173-181). This was also foundto be the case for PorB except purified PorB also functioned in liposomeswelling assays (FIG. 4) and was used in all subsequent experiments. Tocontrol for potential contaminants that may occur during PorBpurification, another predicted outer membrane protein from C.trachomatis serovar D (CT241) was cloned, expressed in E. coli andpurified by the same procedure used for PorB. Like PorB, CT241 alsocontains a predicted leader sequence and ends in phenylalanine and wasincorporated into liposomes and tested for pore forming activity. Thisprotein as well as liposomes without protein did not show pore-formingactivity with any of the solutes tested.

The smallest sugars tested in the liposome swelling assay were themonosaccharides arabinose and glucose. These sugars penetrated the PorB-and MOMP-containing liposomes faster than the disaccharide, sucrose,while the tetrasaccharide, stachyose, was too large to enter (FIG. 4).This diffusion selectivity of PorB- or MOMP-containing liposomes withsugars was similar to what has been observed with COMC-containingliposomes (Bavoil, et al. (1984) Infect. Immun. 44:479-485, Wyllie, etal. (1998) Infect. Immun. 66:5202-5207). Larger solutes enter into PorBor MOMP porin slower, suggesting that there is a size restriction ofmolecules that can enter via these porins. However, the liposomescontaining PorB permitted the diffusion of arabinose or glucose at aslower rate than liposomes containing MOMP.

Since Chlamydia have been proposed to utilize amino acids from hostcells (Ossowski et al. (1965) Isr. J. Med. Sci. 1:186-193; Hatch et al.(1982) J. Bacteriol. 150:379-385; Pearce, (1986) Ann. Inst. PasteurMicrobiol. 137A:325-332), diffusion of amino acids through PorB and MOMPwere tested using the liposome swelling assay. MOMP liposomes allow forthe diffusion of all of the amino acids at different rates basedpredominantly on size selectivity and alanine and glycine enter throughMOMP liposomes slightly faster than arabinose (Jones et al. (2000) Gene258:173-181). In contrast, PorB liposomes did not efficiently allow forany of 20 amino acids to enter liposomes including the small amino acidssuch as alanine (FIG. 5). These data indicate that PorB is lessefficient than MOMP as a non-specific porin.

Example 10 Permeability of Solutes Through PorB

PorB was purified by nickel column chromatography and incorporated intoliposomes. Liposomes enriched for MOMP were used to compare thepore-forming activity of PorB. As shown above, PorB porin function,unlike MOMP, is inefficient in the diffusion of amino acids, even aminoacids smaller in molecular weight than arabinose, such as glycine andalanine. MOMP porin activity is detected using only 1 μg of protein(total outer membrane protein) while 6-10 μg of purified PorB is neededto observe comparable porin activity. This suggests that PorB is muchless efficient as a non-specific porin or that the purification processmay have resulted in a less functional protein.

Differences in general pore-forming activity, as well as differences inthe amount present in the chlamydial outer membrane, suggest a uniquerole for each of the porins. The presence of PorB in small amounts isdifficult to understand unless PorB has a role as a substrate-specificporin that is efficient in the uptake of particular classes ofmolecules. RT-PCR analysis and cell staining at various time pointsindicated that this protein is expressed throughout the developmentalcycle. Thus PorB expression is not differentially regulated.

In order to determine if PorB had specificity for any molecule(s), thegenome sequence was studied to determine if the inferred biology ofChlamydia could provide an idea of which molecules Chlamydia might needto obtain from the host. This analysis provided a list of orthologs oftransporters that are important in the translocation of solutes acrossthe inner membrane, including amino acid, polysaccharide, oligopeptide,and dicarboxylate transporters (Stephens et al. (1998) Science282:754-759). Previous analysis of MOMP porin activity showed that aminoacids, mono- and di-saccharide and oligopeptides enter efficientlythrough MOMP (Jones et al. (2000) Gene 258:173-181). However, PorB didnot allow for the efficient entry of either amino acids orpolysaccharides. The presence of an ortholog to an inner membranedicarboxylate transporter, and that Chlamydia appears to have atruncated TCA cycle, suggest that chlamydiae may require exogenousα-ketoglutarate from the host cell. Therefore, the hypothesis thatdicarboxylates could enter through the chlamydial outer membrane wastested by measuring α-ketoglutarate diffusion through the two knownporins, PorB and MOMP.

The liposome swelling assay with PorB and MOMP showed that the diffusionof α-ketoglutarate was more efficient through PorB than MOMP (FIG. 6).No diffusion of α-ketoglutarate was seen with liposomes without protein,as well as liposomes with another chlamydial outer membrane protein(Omp85) that was purified by the same method as PorB. Chlamydial Omp85was used as a control protein that was cloned, expressed in E. coli andpurified by the same method used to purify PorB. E. coli not expressingPorB, which was treated the same way as E. coli expressing PorB, waspurified by nickel column chromatography and the column eluate was usedas a control in all of the assays to verify that no E. coli contaminantswere responsible for the porin activity observed.

One concern with the liposome assays was the possible influence of ionspresent in anionic solutes, such as α-ketoglutarate, that may cause ionfluxes potentially confounding the results of the assay. A liposomeassay to control for the possibility of ion fluxes (Nikaido andRosenberg (1983) J. Bacteriol. 153:241-252) was used to confirm theswelling assay results. Liposomes were made with NAD⁺-imidazole andstachyose to counteract any ion fluxes that may result from the presenceof contaminating ions in the α-ketoglutarate solute used for the assay.This assay confirmed that the results in the initial liposome assayswere not the result of ion fluxes and that oxaloacetate also enteredefficiently through PorB while citrate did not enter (FIG. 7).

An enzyme-linked liposome assay was used to further show that theα-ketoglutarate was entering through PorB. The liposomes were made withα-ketoglutarate dehydrogenase and NAD⁺ inside and washed. The substrate,α-ketoglutarate, was added to the outside of the liposomes and then theliposomes were measured for the formation of NADH by the increase in theO.D.₃₄₀. This shows that α-ketoglutarate entered through PorB unlike thecontrol liposomes which did not allow α-ketoglutarate to enter insideand result in the formation of NADH (FIG. 8).

Example 11 TCA Cycle Molecules Enter Through PorB

Since α-ketoglutarate efficiently entered through PorB, a number ofother TCA cycle intermediates were tested to assess whether this porinwas specific for the α-ketoglutarate substrate. Succinate (andoxaloacetate) enter PorB with similar rates to α-ketoglutarate; however,malate did not enter efficiently (FIG. 9). Citrate did not enter throughPorB.

Example 12 Permeability Specificity Studies With PorB

Since dicarboxylates of the TCA cycle were tested and diffused throughPorB, other molecular analogues were studied to determine the capabilityof PorB to distinguish between related molecules (FIG. 10). A differencein carbon-chain lengths represented by adipate, glutarate, succinate,and malonate did not show marked differences in diffusion compared toα-ketoglutarate, although 6-carbon adipate and 3-carbon malonate enteredthrough PorB at a slightly slower rate. Thus PorB did not discriminatebetween different substrate chain lengths. The effects of small sidegroups using analogues that differed only by specific side groups weretested. For example, α-ketoglutarate and glutarate entered through PorBefficiently, but not glutamate that is similar in structure. Thepresence of the amino group seems to retard the diffusion of glutamateand this likely explains why other amino acids do not enter into PorBefficiently. A comparison of 5-carbon compounds citrate and aconitatewith only the addition of a hydroxyl group to citrate prevented theentry of citrate through PorB. Four-carbon malate and succinate alsodiffer by the presence of a hydroxyl group and the diffusion rate wasretarded for malate. Therefore, PorB can discern between very similarcompounds to allow for specific selectivity, suggesting asubstrate-specific selective porin. These findings show that PorBfacilitates the diffusion α-ketoglutarate and other selectdicarboxylates to enter chlamydial outer membranes efficiently.

Materials and Methods for Examples: 13-18

The following materials and methods were utilized for Examples 13-18 inconjunction to the materials and methods mentioned above, whereappropriate.

Cloning, expression and purification of PorB. The gene encoding PorB(porB) was cloned into the pBAD TOPO-TA® vector and transformed into E.coli TOP10® competent cells (Invitrogen, Carlsbad, Calif.) as describedby Kubo and Stephens (Kubo and Stephens (2000) Mol Microbiol38:772-780). Host cells containing the recombinant PorB plasmid weregrown in Luria-Bertani at 37° C. until an A₆₀₀ of 0.5 was attained.Protein expression was induced by addition of arabinose at a finalconcentration of 0.02%, and the cultures were incubated for anadditional 3 h. PorB was extracted with 1% octylglucoside at 37° C. for1 h followed by dialysis against PBS. The recombinant protein was thenpurified by nickel column purification using the His-Bind® purificationsystem (Novagen, Madison, Wis.).

Preparation of synthetic peptide conjugates. Twenty-five overlappingpeptides representing the entire PorB sequence were synthesized (GenemedSynthesis Inc., South San Francisco, Calif.; Table 2). Stock solutionsof the PorB peptides (designated B1-1 to B5-5) were prepared indistilled water at a final concentration of 1 mg/ml and stored at −20°C. The peptides were coupled to Imject® maleimide activated-KeyholeLimpet Hemocyanin (KLH; Pierce Endogen, Rockford, Ill.) at a 1:1 ratioof peptide to KLH according to the manufacturers instructions. Briefly,1 mg of maleimide-activated KLH was mixed with 1 mg of PorB peptide in afinal volume of 1 ml and incubated for 2 h at room temperature. Theconjugated protein was dialyzed against PBS (pH 7.4) for 3 h with threebuffer changes. Purified peptide-KLH conjugates were stored at −20° C.until used.

Polyclonal immune sera. Human immune sera were obtained from individualsnaturally infected with C. trachomatis. Polyvalent antisera to C.trachomatis serovar B was obtained from rabbits immunized with purifiedEB as previously described (Caldwell, H. D., C. C. Kuo, and G. E. Kenny(1975) J Immunol 115:963-968). Monospecific polyclonal antisera torecombinant PorB and synthetic PorB peptide conjugates were produced inSwiss Webster mice (Harlan, San Diego Calif.). Five 6-8 week old femalemice were immunized by subcutaneous injection with 15 μg of purifiedPorB protein or 100 μg peptide-KLH conjugate in an equal volume ofComplete Freund's Adjuvant. Intraperitoneal boost immunizations wereperformed two weeks later in Incomplete Freund's Adjuvant. After anadditional two weeks, mice were tested for reactivity to homologouspeptide by peptide-specific ELISA. Institutional Review Board approvalwas obtained for use of human sera and immune sera production in rabbitsand mice.

Peptide-specific ELISA. Mouse immune sera to PorB peptides were screenedby ELISA using homologous peptide as coating antigen. Polystyrenemicrotiter plates (IMMULON 2; Dynatech, Chantilly, Va.) were coated with50 μl of 5 μg/ml of peptide per well in 50 mM bicarbonate buffer (pH9.6) and incubated overnight at 37° C. After washing twice withPBS-Tween (PBS; 0.05% TWEEN 20), the wells were incubated with 100 μlblocking buffer containing 2% gelatin in PBS for 1 h at 37° C. and thenwashed with PBS-Tween. A 50 μl volume of a 1:1000 dilution of the mouseantisera was added and the plates incubated for 1 h at 37° C. The wellswere washed three times with PBS-Tween, and incubated with a 1:2000dilution of goat anti-mouse IgG-horseradish peroxidase (HRP) conjugatedantibody (Zymed laboratories, South San Francisco, Calif.) for 1 h at37° C.

After washing three times in PBS-Tween and twice in PBS, the antibodycomplexes were detected with a mixture of substrate (0.1% hydrogenperoxide) and chromogen (1 mg/ml o-phenelynediamine; Dako Corporation,Carpinteria, Calif.) in 0.1 M citrate buffer. The color was allowed todevelop for 15 min and the reaction was terminated by addition of 25 μlof 8N H₂SO₄. The absorbance at 492 nm was measured on a TitertekMultiscan

ELISA plate reader (Flow Laboratories, McLean, Va.). Each assay was runin duplicate. Human and rabbit immune sera were tested for reactivity toPorB peptides in a similar ELISA format at 1:1000 dilutions. Sampleswere treated with either goat anti-human IgG-HRP or goat anti-rabbitIgG-HRP conjugates (Zymed laboratories, South San Francisco, Calif.) assecondary antibody and binding to PorB peptide was detected as describedabove.

Surface accessibility ELISA. The ability of PorB peptide antisera torecognize their cognate epitopes on the surface of viable chlamydiae wasdetermined by absorption ELISA. Peptide antisera were adjusted to adilution corresponding to a A₄₉₂ range of 0.4 to 1.8 and pre-incubatedfor 30 min at room temperature with purified chlamydial EB (˜10⁸IFU/ml). After centrifugation to remove the EB, the peptide antiserawere tested for residual reactivity to homologous PorB peptide orrecombinant PorB as described in the ELISA method above. The differencein reactivity (A₄₉₂) between the absorbed and unabsorbed peptideantisera was calculated and statistically analyzed by a Student t-test.The absorption experiments were repeated twice.

Chlamydial dot-blot assay. The dot-blot assay was performed aspreviously described by Zhang et al (Zhang, Y. X. et al (1987) J Immunol138:575-581). Briefly, nitrocellulose membrane (Bio-Rad Laboratories,Hercules, Calif.) was presoaked in PBS for 10 min and assembled onto adot-blot apparatus (Bio-Rad Laboratories, Hercules, Calif.). A 50 μlsuspension of chlamydial EB (5 μg/ml) in PBS was added to appropriatewells and was allowed to filter by gravity for 10 min followed by vacuumfiltration for 5 min to remove all liquid from the wells. The membranewas removed and treated with a blocking solution (2% dried skim milk inPBS) on a rocker for 1 h at room temperature. After three washes inPBS-Tween, the membrane was re-assembled. Dilutions of peptide antiserawere added to corresponding wells and incubated for 1 h at roomtemperature.

After the wells were washed three times with 200 μl PBS-Tween, themembrane was removed from the blotting apparatus and washed three moretimes with PBS-Tween. A 1:2000 dilution of goat anti-mouse IgGHRP-conjugate was added to the membrane as secondary antibody andincubated for 1 h at room temperature. After three washes in PBS-Tween,the membrane was treated with enhanced chemiluminescence (ECL) detectionreagents (Amersham Pharmacia Biotech, Piscataway, N.J.) and subjected toautoradiography for 5 sec using Kodak X-omat AR film (Eastman Kodak Co.,Rochester, N.Y.). Controls included probing immobilized EB with (a) 2C5,a monoclonal antibody to the species-specific VS4 region of MOMP, (b)pre-immune sera and (c) anti-Pgp3, an inner membrane chlamydial protein.

In vitro neutralization. In vitro neutralization assays using HaK(Syrian Hamster Kidney) cells were performed as previously described(Byrne, G. I., R. S. Stephens, and et al. (1993) J Infect Dis168:415-420). Serial dilutions of monospecific PorB peptide antisera,were prepared in SPG. Mouse antisera produced to full-length PorB wasused as positive control and pre-immune sera and SPG were used asnegative controls. C. trachomatis serovar D EB were diluted in SPG tocontain 2×10⁴ inclusion forming units (IFU)/ml and 90 μl were added toeach serum dilution in a final volume of 180 μl. Neutralization wasallowed to proceed for 30 min at 37° C. 50 μl of each sample were addedin triplicate to PBS-washed HaK monolayers and incubated for 2 h at 37°C. After excess inoculum was removed, the cells were rinsed once withPBS, replenished with 200 μl of RPMI medium supplemented with 10% fetalbovine serum and 1 μg/ml cycloheximide, and incubated at 37° C. for 48h. Chlamydial inclusions were detected by staining with mouse anti-C.trachomatis MOMP fluorescent antibody (Wampole Laboratories, Cranbury,N.J.) and quantified by counting 3 fields per well at a magnification of40×. The results were calculated as percentage reduction in mean IFUrelative to the control SPG. Assays were performed in triplicate.

Example 13 Epitope Mapping With Immune Sera

The first step in elucidating the structure of PorB was to determinewhich regions of the protein are antigenic. To define antigens of PorB,the binding of human immune sera, rabbit anti-EB sera and mouseanti-PorB sera to 25 synthetic overlapping peptides spanning the PorBsequence (Table 2) was tested by peptide-specific ELISA. Peptides withhigh frequencies and high titers of reactivity to the differentpolyclonal immune sera were considered immunoreactive.

Using these criteria, four major neutralizing epitope clusterscorresponding to Phe₃₄-Leu₅₉ (B1-2 and B1-3), Asp₁₁₂-Glu₁₄₅ (B2-3 andB2-4), Gly₁₇₉-A₂₂₅ (B3-2, B3-3 and B3-4) and Val₂₆₁-Asn₃₀₅ (B4-4, B5-1and B5-2) were identified (FIG. 11). Each neutralizing epitope domaincomprised 2-3 epitopes, with higher peaks of reactivity noted forindividual peptides B1-3, B2-3, B2-4, B3-4 and B5-2 for all sera tested.The C-terminal end of the protein appeared as a major immunoreactiveregion with two epitope clusters in close proximity. Although variationsin individual reactivities exist, all antisera recognized the samedeterminants, indicating that these epitopes are broadly antigenic. Eachof the individual sera showed reactivity to purified PorB antigen in asimilar ELISA format, confirming that antibody recognition of the PorBpeptides was specific (data not shown). Except for peptide B2-3 thatcontained a stretch of hydrophobic residues in its C-terminal region,most of the reactive epitopes were hydrophilic. The hydrophilicity andreactivity of the immunoreactive epitopes suggest that they may besurface-accessible and targets of neutralizing immune responses.

TABLE 2 Synthetic Peptides Representing PorB Amino Acid sequence^(a)PEPTIDE SEQUENCE SEQ ID NO. B1-1 LDAMPAGNPAFPVIPG^(b) (SEQ ID NO. 35)B1-2 FPVIPGINIEQKNACS (SEQ ID NO. 11) B1-3 QKNACSFDLCNSYDVL (SEQ ID NO.12) B1-4 NSYDVLSALSGNLKLC (SEQ ID NO. 21) B1-5 GNLKLCFCGDYIFSEE (SEQ IDNO. 22) B1-6 YIFSEEAQVKDVPVVT (SEQ ID NO. 23) B2-1 DVPVVTSVTTAGVGPSPDIT(SEQ ID NO. 24) B2-2 PSPDITSTTKTRNFDLVNCN (SEQ ID NO. 25) B2-3DLVNCNLNTNCVAVAFSLPD (SEQ ID NO. 13) B2-4 AFSLPDRSLSAIPLFDVSFE (SEQ IDNO. 14) B2-5 FDVSFEVKVGGLKQYYRLP (SEQ ID NO. 26) B2-6QYYRLPMNAYRDFTSEPLNS (SEQ ID NO. 27) B3-1 TSEPLNSESEVTDGMIEVQS (SEQ IDNO. 28) B3-2 GMIEVQSNYGFVWDVSLKKV (SEQ ID NO. 15) B3-3DVSLKKVIWKDGVSFVGVGAD (SEQ ID NO. 16) B3-4 FVGVGADYRHASCPIDYIIA (SEQ IDNO. 17) B4-1 PIDYIIANSQANPEVFIADS (SEQ ID NO. 29) B4-2VFIADSDGKLNFKEWSVCVG (SEQ ID NO. 30) B4-3 WSVCVGLTTYVNDYVLPYLA (SEQ IDNO. 31) B4-4 VLPYLAFSIGSVSRQAPDDSF (SEQ ID NO. 18) B5-1APDDSFKKLEDRFTNLKF (SEQ ID NO. 19) B5-2 FTNLKFKVRKITSSHRGN (SEQ ID NO.20) B5-3 SSHRGNICIGATNYVADN (SEQ ID NO. 32) B5-4 NYVADNFFYNVEGRWGSQ (SEQID NO. 33) B5-5 GRWGSQRAVNVSGGFQF (SEQ ID NO. 34) ^(a)Residuesrepresenting overlapping peptide regions are underlined. All peptidesexcept B1-4 were synthesized with an additional cysteine at their Ctermini for conjugation to KLH. ^(b)Residues are listed in single-lettercode beginning at the N-terminal end.

Example 14 Determination of Surface Accessibility of PorB Epitopes

Neutralization of chlamydial infectivity requires that antigenicdeterminants are surface exposed and accessible for antibody recognition(Fan, J., and R. S. Stephens 1997) J Infect Dis 176:713-721; Toye, B.,G. M. Zhong, R. Peeling, and R. C. Brunham (1990) Infect Immun58:3909-3913). The experimental approach to determining whether theidentified PorB epitopes are surface-accessible was to generate a panelof mouse antisera to each of 25 overlapping synthetic peptidesrepresenting PorB (Table 2), and to use these to probe for their cognateantigen on chlamydial EB in absorption ELISA and EB surface-specificdot-blot assays.

Characterization of Anti-peptide Conjugate Antibodies.

A panel of mouse antisera raised to overlapping synthetic PorB peptideswas evaluated for reactivity by a peptide-specific ELISA assay. All serareacted with their homologous peptides with mean end-point titers (log₂values) ranging from 9 to 18 (Table 2). The reactivity ofpeptide-specific antisera to recombinant PorB or homologous peptides wasreduced 58 to 95% by competitive inhibition with respective peptide,demonstrating that peptide recognition was specific (data not shown). ).In contrast, the peptide sera were unaffected by absorption with KLH andan unrelated chlamydial peptide Ct673 (data not shown). Since MOMPquantitatively predominates the surface of chlamydial EB and is known tomediate neutralization of infectivity, the panel of peptide antisera wastested for cross reactivity to this protein. No cross-reactivity of thepeptide-specific antisera was observed as reactivity to recombinant MOMPprotein was <0.3 A₄₉₂ for all sera tested (Table 2).

EB-Absorption Studies

To determine which of the peptide-specific antibodies were directed atPorB epitopes on the chlamydial EB surface, absorption studies wereconducted in an ELISA format. Peptide-specific antisera werepre-incubated with viable EB prior to testing for reactivity to PorBpeptides and recombinant PorB. If absorption with intact EB reduced thereactivity of peptide-specific antibody compared to the unabsorbed sera,it can be inferred that the absorbed antibodies were directed towardsurface-accessible epitopes on PorB. Absorption of sera raised toepitopes B1-2, B1-3, B2-1, B2-3, B2-4, B3-2, B3-4, and

B5-2 with chlamydial EB resulted in a significant decrease in reactivityto their cognate peptides (FIG. 12; P<0.05). Antisera B2-3, B3-2, B3-4and B5-2 showed the largest decreases in reactivity when absorbed withEB. In contrast, antisera B1-6 and B2-6, which showed high reactivitywith their respective peptides (Table 3), showed no decreases inreactivity when absorbed with EB. This verified that the chlamydial EBwere intact during absorption. Reactivity of peptide-absorbed antiserato recombinant PorB revealed similar profiles of surface-exposedepitopes indicating that the peptide-specific antibodies bind to cognatepeptides as well as whole protein (data not shown). Likewise, whenpolyclonal immune sera from humans, rabbits and mice were pre-absorbedwith EB and tested for reactivity with PorB peptides, a decrease inreactivity was observed for the same epitopes (FIG. 13). The data fromEB-absorption studies supported the specificity of the peptide-antibodyinteraction and provided evidence that the immunoreactive PorB epitopesare surface-exposed.

TABLE 3 Immunological properties of mouse antisera raised against PorBpeptide conjugates Cross-reactivity Homologous peptide with rMOMPAntibody ELISA reciprocal titer (log₂)^(a) ELISA (A₄₉₂)^(b) B1-1  15^(c) 0.24 B1-2 13 0.18 B1-3 13 0.09 B1-4 15 0.03 B1-5 16 0.19 B1-616 0.00 B2-1 13 0.00 B2-2 15 0.30 B2-3 12 0.00 B2-4 14 0.00 B2-5 15 0.04B2-6 17 0.27 B3-1 12 0.21 B3-2 12 0.16 B3-3 11 0.07 B3-4 17 0.22 B4-1 110.14 B4-2 11 0.01 B4-3 13 0.27 B4-4 14 0.19 B5-1 11 0.11 B5-2 13 0.02B5-3 11 0.19 B5-4  9 0.02 B5-5 11 0.00 Anti-rMOMP^(d) n/a 2.31^(a)Serial two-fold dilutions of peptide antisera were made and testedfor reactivity to their homologous peptides at 5 μg/ml. Absorbancevalues were subtracted from background (range 0.05–0.160). ^(b)Peptideantisera were diluted 1:1000 and tested for reactivity to recombinantMOMP. Absorbance values (range 0.05–0.160) were subtracted frombackground. ^(c)Groups of 5 mice were immunized for each peptideconjugate. The data shown represent mice with the highest antibodytiter. ^(d)Anti-rMOMP sera was obtained from mice immunized withrecombinant MOMP and tested at 1:1000 for reactivity to relevant antigenat 5 μg/ml.Dot-Blot Analyses

To confirm the specificity and surface reactivity of the PorB peptideantisera, a dot-blot analysis was performed with chlamydial EB aspreviously described by Zhang et al. (Zhang, Y. X. et al (1987) JImmunol 138:575-581). Anti-B2-1, B2-3, B3-4 and B5-2 showed thestrongest reactivities to chlamydial EB, followed by anti-B1-2, B1-3,B2-4 and B4-2 (FIG. 14). This demonstrates that the cognate epitopes forthese antibodies are exposed on the bacterial surface.

Epitope B3-2, that showed surface accessibility by the EB-absorptionmethod, did not show a strong signal in the dot-blot assay. In contrast,epitope B4-2 that was not surface-exposed by EB-absorption gave a strongsignal by the dot-blot method. The absence of a reactive signal for theremaining PorB peptide antisera implies that their respective epitopesare inaccessible for antibody recognition.

When the EB were treated with SDS, the dot-blot profile for all thepeptide antisera was very strong, indicating that SDS had solubilizedthe outer membrane of the EB and all PorB epitopes were now accessible(FIG. 14). Pre-immune sera were negative for reactivity with bothSDS-treated and untreated EB. An additional control was included inwhich sera to a non-surface protein, Pgp3 (Comanducci, M. et al (1993) JGen Microbiol 139:1083-1092) was used to probe immobilized EB. Thisantibody did not bind to the intact EB but showed strong reactivity withlysed EB, verifying the structural integrity of the viable EB. Thefindings in the dot-blot analyses were consistent with those of theEB-absorption studies and confirmed that the major antigenicdeterminants of PorB are surface-exposed.

With the exception of two epitopes, the absorption and dot-blot studieswith peptide antisera consistently identified the same seven epitopes assurface-exposed (FIG. 16). The discrepancy observed for B3-2 and B4-2may be attributed to fundamental differences between the ELISA and thedot-blot methods or differences in the peptide-specific antibodypopulations being recognized. Nevertheless, when polyclonal immune serafrom humans and rabbits were used in similar EB-absorption studies, bothepitopes were recognized as surface-accessible.

Example 15 In vitro Neutralization of Chlamydial Infectivity

In vitro neutralization assays are a central component in evaluatingfunctionality of chlamydial immune responses and provide the bestcorrelate of protective immune responses (Byrne, G. I., R. S. Stephensand et al (1993) J Infect Dis 168:415-420). The neutralization data forthe peptide antisera revealed four regions of neutralizing activity onthe PorB protein with 50% reciprocal neutralization end-point titersranging from about 32 to about 2048 (FIG. 15). Anti-B1-3, B2-4 and B5-2sera provided the strongest neutralization activities with 50%reciprocal end-point titers >1024 followed by anti-B2-3, B3-2, B3-4 andB4-2. Notably, these regions of neutralizing activity overlap regionsidentified as immunoreactive and surface-exposed by ELISA and dot-blotanalysis. This confirms that the epitopes contributing to theneutralizing property of PorB antisera are surface-exposed.

When a pool of peptides representing the strongly neutralizing epitopes(B1-3, B2-3 B2-4, B3-2, B3-4 B4-2 and B5-2) was used in a competitiveinhibition assay with PorB antisera, the neutralizing ability of PorBantisera was markedly reduced (Table 4). Inhibition of neutralizationwas concentration-dependent with 10 μg of peptides completely blockingneutralization. Similarly, pre-incubation of the PorB antisera withpurified recombinant PorB also ablated neutralization of chlamydialinfectivity. Heat-denaturation of PorB appeared to have no additionaleffect on inhibiting PorB antisera since the neutralization results weresimilar to that observed when intact PorB was used as the inhibitingantigen. These results revealed that neutralizing PorB antisera containa population of antibodies that recognize predominantly linear epitopeson the surface of the EB.

TABLE 4 Percent Neutralization of chlamydial infectivity by PorBantisera in the presence of inhibitory antigen Concentration (μg)^(a)Antigen 2 5 10 Peptides^(b)   28%^(c) 0% 0% PorB 39% 11%  0% DenaturedPorB^(d) 39% 6% 11%  ^(a)Increasing concentrations of inhibiting antigenwere pre-incubated with a dilution of PorB antisera yielding ~60%neutralization and used in in vitro neutralization assays as describedin the methods section. ^(b)A pool of synthetic peptides representingepitopes that are neutralizing targets were used for inhibition studies.^(c)% Neutralization was calculated for each sample relative to the SPGcontrol. Assay was done in triplicate. ^(d)Purified PorB protein wasdenatured by boiling for 10 minutes.

The neutralization profile developed with PorB peptide antisera revealsfour major regions of neutralizing activity that overlap the regionsidentified as immunoreactive and surface-exposed (FIG. 16). Theproximity of the neutralizing epitopes supports the existence of acomplex of discontinuous antigenic structures that contribute to thePorB-specific neutralizing immune response. Since antibodies raised tosynthetic peptides may be limited in their recognition of linear versusconformational epitopes on the native antigen, caution must be exercisedin interpreting the PorB neutralization data. Although the complex ofdiscontinuous antigenic structures as defined by the peptide-specificantisera does not exclude the presence of conformational epitopes,evidence provided in this study that the neutralizing activity of PorBantisera is ablated by synthetic PorB peptides or purified PorB suggeststhe presence of a large population of PorB antibodies whose neutralizingactivity is independent of conformational requirements.

Example 16 Identification of Common PorB Epitopes Between C. trachomatisand C. pneumoniae

Comparison of the PorB amino acid sequence for C. trachomatis and C.pneumoniae revealed 59.3% identity, indicating that this protein isconserved between species (Kubo and Stephens (2000) Mol Microbiol38:772-780). In order to determine whether the neutralizing C.trachomatis PorB peptide antisera recognized common epitopes between thetwo species, the reactivity of the neutralizing PorB peptide antisera toanalogous synthetic C. pneumoniae PorB peptides was evaluated. Four C.pneumoniae PorB peptides (Cpn 1-3, Cpn 3-2, Cpn 4-4 and Cpn 5-2) showedstrong reactivities to the corresponding C. trachomatis peptide antisera(Table. 5). Amino-acid sequence comparison revealed that C. pneumoniaePorB peptides with the highest cross-reactivity (Cpn 3-2, Cpn 4-4 andCpn 5-2) shared a minimum of 67% residue identity (bold) and 75%sequence similarity with their corresponding C. trachomatis PorBpeptides. In contrast peptides with less than 50% sequence identity(Cpn1-2, Cpn2-3, Cpn2-4) showed little or no cross-reactivity with thecorresponding peptide antisera.

Antibody recognition of Cpn PorB epitopes was not always associated withan increased number of shared or similar residues. For instance, Cpn 4-3which has 60% identity and 90% similarity with B4-3 showed no reactivitywith the corresponding B4-3 antisera, whereas, Cpn2-4 with 40% identityand only 50% similarity showed moderate cross-reactivity. The reactivitypatterns suggest that these C. pneumoniae epitopes may be surfaceexposed and may also be the targets of neutralizing antibody responses.Collectively, these results show that neutralizing PorB peptide antiserarecognize similar PorB epitopes in C. trachomatis and C. pneumoniae andsuggest that the sera may have broad neutralizing properties directedagainst surface-exposed epitopes.

TABLE 5 Reactivity of neutralizing C. trachomatis PorB peptide-specificantisera to synthetic C. pneumoniae PorB peptides Reactivity to peptideantisera Peptide Amino-acid sequence^(a) ELISA (A₄₉₂)^(b) Cpn 1-2 A PV LPG V N P EQ TGWCA (SEQ ID NO.36) 0.10 Cpn 1-3 Q TGWCAFQ LCNSYD LF (SEQID NO.37) 1.24 Cpn 2-3 DL N N SSISSS CV FATIA L QE (SEQ ID NO.38) 0.49Cpn 2-4 TIA L QET S PA AIPL LDIA F T (SEQ ID NO.39) 0.58 Cpn 3-2 G LIEVQS D YG I VW GL SL Q KV (SEQ ID NO.40) 2.76 Cpn 3-3 GL SL Q KV LWKDGSFVGV S AD (SEQ ID NO.41) 0.19 Cpn 3-4 FVGV S ADYRH G S S PI N YII V(SEQ ID NO.42) 0.50 Cpn 4-2 IYFDAT DG N L SY KEWS ASIG (SEQ ID NO.43)0.82 Cpn 4-3 WS ASI G IS TY L NDYVLPY AS (SEQ ID NO.44) 0.12 Cpn 4-4VLPY ASV SIG NT SR K AP S DSF (SEQ ID NO.45) 3.00 Cpn 5-2 FTN F KFK IRKIT NFD R V N (SEQ ID NO.46) 2.08 ^(a)Synthetic peptides analogous toC. trachomatis PorB peptides were synthesized as described in themethods section. Residues are listed in single-letter code beginning atthe N-terminal end. Regions of shared amino-acid sequences are in boldand underlined. ^(b)Peptide-specific antisera were diluted 1:1000 andtested for reactivity to C. pneumoniae PorB peptides at 5 μg/ml.Absorbance values at 492 nm were compared to values for reactivity ofantisera to cognate C. trachomatis peptides.

Identification of the major neutralizing epitopes for PorB has importantimplications for chlamydial vaccine design. In addition to havingsurface-accessible and possibly conformation-independent antigenicdeterminants, a major advantage of PorB is that it is highly sequenceconserved between serovars and species (Kubo and Stephens (2000) MolMicrobiol 38:772-780) and can be expected to provide protection for abroad spectrum of chlamydial strains including C. pneamoniae. Consistentwith this, is the finding that neutralizing antibodies to C. trachomatisserovar D PorB epitopes cross-react with analogous C. pneumoniaepeptides and also neutralize infectivity of C. trachomatis serovar B(data not shown). Moreover, the C. pneumoniae PorB peptides used in thisstudy have similar hydrophilicity profiles as their C. trachomatiscounterparts, increasing the likelihood that these epitopes are alsosurface oriented on the native antigen and are targets of a broadlyneutralizing antibody response. PorB as a vaccine antigen will obviatethe need to incorporate serovar and species-specific determinants and ifeffective, will be valuable in providing protection against multipleserovars or species, which is highly desirable for long-term control ofchlamydial infections.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. An isolated nucleic acid comprising a nucleic acid sequence encodinga C. pneumoniae PorB polypeptide up to 60 amino acids in length whereinthe polypeptide contains an amino acid sequence that is at least 67%identical to the amino acid sequence FTNLKFKVRKITSSHRGN (SEQ ID NO: 20),wherein the polypeptide binds to neutralizing C. trachomatis PorBpolypeptide-specific antisera.
 2. The isolated nucleic acid of claim 1,wherein the polypeptide has the amino acid sequence ofFTNLKFKVRKITSSHRGN (SEQ ID NO: 20).
 3. The isolated nucleic acid ofclaim 1, wherein the polypeptide has the amino acid sequence ofVLPYLAFSIGSVSRQAPDDSFKKLEDRFTNLKFKVRKITSSHRGN (SEQ ID NO: 10).
 4. Theisolated nucleic acid of claim 1, wherein the polypeptide comprises atleast 30 amino acids.
 5. The isolated nucleic acid of claim 1, whereinthe polypeptide comprises at least 20 amino acids.
 6. The isolatednucleic acid of claim 1, wherein the polypeptide comprises at least 15amino acids.
 7. The isolated nucleic acid of claim 1, wherein thepolypeptide has the amino acid sequence of FTNFKFKIRKITNFDRVN (SEQ IDNO: 46).
 8. An isolated nucleic acid comprising a nucleic acid sequenceencoding a C. pneumoniae PorB polypeptide up to 60 amino acids in lengthhaving the amino acid sequence of FTNLKFKVRKITSSHRGN (SEQ ID NO: 20). 9.The isolated nucleic acid of claim 8, wherein the polypeptide has theamino acid sequence of FTNLKFKVRKITSSHRGN (SEQ ID NO: 20).
 10. Theisolated nucleic acid of claim 8, wherein the polypeptide has the aminoacid sequence of VLPYLAFSIGSVSRQAPDDSFKKLEDRFTNLKFKVRKITSSHRGN (SEQ IDNO: 10).
 11. The isolated nucleic acid of claim 8, wherein thepolypeptide comprises at least 30 amino acids.
 12. The isolated nucleicacid of claim 8, wherein the polypeptide comprises at least 20 aminoacids.